Radiolabeled ligands for targeted pet/spect imaging and methods of their use

ABSTRACT

The present disclosure provides compounds, complexes, compositions, and methods for the detection of cancer. Specifically, the compounds, complexes, compositions of the present technology include pH (low) insertion peptides. Also disclosed herein are methods of using the complexes and compositions of the present technology in diagnostic imaging to detect cancer in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ApplicationNo. 62/368,991, filed Jul. 29, 2016, the contents of which areincorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under CA186721,CA138468, and CA008748, awarded by the National Institutes of Health.The government has certain rights in the invention.

TECHNICAL FIELD

The present technology relates generally to compositions including amodified pH (low) insertion peptide and methods of using the same indiagnostic imaging to detect acidic tissues, such as cancer, in asubject.

BACKGROUND

The following description of the background of the present technology isprovided simply as an aid in understanding the present technology and isnot admitted to describe or constitute prior art to the presenttechnology.

Cancer cells generally prefer the glycolytic pathway of energyproduction due to their deregulated proliferative machinery and survivalneeds, thus resulting in excess acidity. To maintain homeostasis, cancercells release lactic acid formed during glycolysis to the extracellularenvironment. Lactic acid release lowers the extracellular pH immediatelysurrounding these cells relative to the pH of normal tissues. Due to thedifferential in pH gradients, a probe marking these acidic regions inrapidly proliferating tissues can potentially discriminate canceroustissues from normal tissue.

Thus, there is a need for novel diagnostic compositions that exhibit (a)high tumoral uptake and prolonged retention in tumors, and (b) minimalaccumulation in non-tumor tissue for use in PET and SPECT imagingmethods to detect tumor cells.

SUMMARY OF THE PRESENT TECHNOLOGY

In one aspect, the present disclosure provides a compound orpharmaceutically acceptable salt thereof, where the compound includes apH (low) insertion peptide (“pHLIP”) configured to localize to anextracellular environment having a pH that is lower than 7.4, whereinthe pHLIP includes a C-terminus and an N-terminus; and X¹ covalentlyattached to a heteroatom of a side chain of an amino acid residue of thepHLIP, where the amino acid residue is from 0, 1, 2, 3, 4, 5, or 6residues from the C-terminus or the N-terminus.

In the composition, X¹ is of Formula I

where Z¹ and Z² are each independently a lone pair of electrons (i.e.providing an oxygen anion) or H; and Z³ is —NH-alkylene-,—NH—CH₂CH₂—((poly(alkylene glycol))-,

where n is 1, 2, 3, 4, 5, 6, 7, or 8, or a bond.

In a related aspect, a complex is provided that includes any embodimentof the composition described herein and a radionuclide. In such acomplex, X¹ may be of Formula II

wherein M¹ is ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺,⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.

In another related aspect of the present technology, a composition isprovided that includes any one of the aspects and embodiments ofcompounds and/or complexes and a pharmaceutically acceptable carrier. Ina further related aspect, a pharmaceutical composition is provided, thepharmaceutical composition including an effective amount of any one ofthe embodiments of the complexes described herein for imaging a tissuecomprising an extracellular environment having a pH that is lower than7.4 and a pharmaceutically acceptable carrier.

In an aspect, the present disclosure provides a method for detectingsolid tumors in a subject in need thereof comprising (a) administeringto the subject an effective amount of a complex of any embodimentdescribed herein, wherein the complex is configured to localize to asolid tumor having an acidic pH environment; and (b) detecting thepresence of solid tumors in the subject by detecting radioactive levelsemitted by the complex that are higher than a reference value.

In another aspect, the present disclosure provides a method fordetecting acidic diseased tissue in a subject in need thereof comprising(a) administering to the subject an effective amount of a complex of anyembodiment described herein, wherein the complex is configured tolocalize to an extracellular environment having a pH that is lower than7.4; and (b) detecting the presence of acidic diseased tissue in thesubject by detecting radioactive levels emitted by the complex that arehigher than a reference value.

Also disclosed herein are kits containing components suitable fordiagnosing, e.g., cancer, in a patient. In one aspect, the kits compriseat least one compound or complex of the present technology, instructionsfor use, and optionally at least one radionuclide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the three folding states of pH (low) insertion peptides(“pHLIPs”) in decreasing extracellular pH: unfolded and unbound peptidein solution (State I), unfolded peptide loosely interacting with themembrane lipid bilayer at physiological pH (State II), and foldedpeptide in alpha-helical conformation inserted across the membrane atlow extracellular pH (State III).

FIG. 2A shows the chelator NOTA-pHLIP. FIG. 2B shows the chelatorNO2A-cyspHLIP where the NO2A chelator is attached to the pHLIP through acysteine at the N-terminus. FIG. 2C shows the chelator NO2A-cyspHLIPwhere the NO2A chelator is attached to the pHLIP through a cysteine atthe C-terminus. The chiral centers within the chelator and linker aremarked with asterisks.

FIG. 3A shows coronal PET image slices at the level of the tumor at 4hours post injection (h. p.i.) (FIG. 3A). FIG. 3B and FIG. 3C show exvivo biodistributions in selected organs at 4 and 24 h p.i.respectively, which highlight differences in the distribution ofcomplexes (alteratively referred to “tracers” or “tracer compounds”) inBALB/c female mice bearing 4T1 tumor allografts. The arrowheads in FIG.3A indicate where the tumor is located in the mouse. The selected scaleon the PET images does not allow for tumor visualization in the case ofthe NO2A-cysVar7 complexes. FIG. 3B shows the maximum intensityprojections (MIPs) for 12 tracer compounds at 4 h and the slices at thelevel of the tumor for the [¹⁸F]—AlF-NO2A-cys pHLIP complexes with amaximum value of 15% ID/g. Only ex vivo biodistribution data wasobtained for ⁶⁴Cu-NOTA-Var7.

FIGS. 4A and 4B show pH dependent bilayer insertion of NO2A-cysVar3complexes. Changes in intrinsic complex fluorescence are used to measurethe insertion of the complex population as a function of pH (transitionfrom State II, at high pH, to State III, at low pH). FIG. 4A: Amount ofcomplex population in State II is measured on the y-axis. 95% confidenceintervals are indicated by yellow bands. FIG. 4B: pH-dependenceparameters are used to calculate the percent of inserted complexpopulation at various pH levels. These transitions were measured in thepresence of physiological levels of free magnesium and calcium ions(0.65 mM and 1.25 mM, respectively).

FIG. 5 shows slices and maximum intensity projections (MIP) from PETimages showing differences in the radiolabeled NO2A-cysVar3 distributionat 4 and 24 h in BALB/c female mice bearing orthotopic 4T1 tumorallografts, nude male mice bearing shoulder PC3 or LNCaP xenografts, andC57Bl/6 female mice bearing orthotopic B16-F10 tumor allografts. Thearrowheads indicate where the tumor is located in the mouse.

FIG. 6 shows a graph of the ex vivo tumor uptake of the leadradiolabeled NO2A-cysVar3 in various tumor models at 4, 6, and 24 h p.i.

FIG. 7 shows a comparison of uptake in sections of excised tumors fromPC3 or LNCaP tumor-bearing male nude mice. The top panels are expansionsof the whole tumor sections shown below. The left-most panels are H&Estained, the middle panels are the autoradiography, and the right-mostpanels are overlays.

FIGS. 8A and 8B show PET Maximum intensity projection (MIP) showingdifferences in the tracer distribution at 4 h p.i. for all 12 tracers(FIG. 8A) and the PET imaging slices at 15% ID/g at 4 h p.i. for the[¹⁸F]—AlF-cyspHLIP complexes in BALB/c female mice bearing 4T1 tumorallografts (FIG. 8B). The arrowheads indicate where the tumor is locatedin the mouse. The MIPs correspond to the same images shown in FIG. 3A.The slices are the same as those shown in FIG. 3A, just at 15% ID/g.Only ex vivo biodistribution data was obtained for ⁶⁴Cu-NOTA-Var7.

FIG. 9 shows three states of NO2A-cysVar3 and NOTA-Var3 complexes. Thecomplexes were examined under varying conditions for the presence of thethree states of pHLIP: State I is the complex in solution at pH 8 (blacklines), State II is the complex in the presence of POPC liposomes at pH8 (blue lines), and State III is the complex folded and inserted in thelipid membrane when the pH is dropped from pH 8 to pH 4 by the additionof an aliquot of HCl (red lines). The concentrations of complex andlipids were 5 μM and 1 mM, respectively.

FIG. 10 shows State II and State III of NO2A-cyspHLIP complexes. Thecomplexes were examined in the presence of liposomes under varying pHlevels: State II is the complex in the presence of POPC liposomes at pH8 (blue lines), State III is the complex folded and inserted in thelipid membrane when the pH is dropped from pH 8 to pH 4 by the additionof an aliquot of HCl (red lines). The concentrations of complex andlipids were 5 μM and 1 mM, respectively, with physiological levels offree magnesium and calcium ions (0.65 mM and 1.25 mM, respectively).

FIG. 11 shows pH dependent bilayer insertion of complexes. Changes inintrinsic complex fluorescence are used to measure the insertion of thecomplex population as a function of pH (transition from State II, athigh pH, to State III, at low pH). The amount of complex population inState II is measured on the y-axis. The red and blue lines are fittingcurves and 95% confidence intervals, respectively. These experimentswere carried out with physiological levels of free magnesium and calciumions (0.65 mM and 1.25 mM, respectively).

FIGS. 12A and 12B show initial results from the brain tumor uptake studydisclosed herein. FIG. 12A shows the images of the uptake of U87MGtumors xenografted in the skull (U87MG*) or flank (U87MG) on male nudemice and GSC 5-22 tumors orthotopically xenografted behind an intact BBBin IRC-SCID male mice at 4 h post injection. The U87MG* tumors did notinfiltrate the brain and only the portion of the tumor that was in theskull bone and protruding from the skull showed uptake. The arrowheadsin FIG. 12A indicate where the tumor is located in the mouse. FIG. 12Bshows the complex biodistribution of six male nude mice with flank U87MGxenografted tumors.

FIG. 13 shows the relative autoradiography of tumor slices from FIG. 7.The slices show the overall difference in uptake of the tracer at 24 hp.i. Both of the slices were on the same autoradiography plate and,therefore, are relative to each other.

FIGS. 14A and 14B show a comparison of tumor uptake to previouslyreported studies for PC3 (FIG. 14A) and LNCaP (FIG. 14B) tumor bearingnude male mice at 1, 4, and 24 h with ex vivo biodistribution and invivo imaging values. For imaging time points, ROIs were drawn on thecoronal, sagittal, and transverse slices and the middle median value wastabulated for each mouse, the average of the four mice are shown withthe standard deviation. Values from previous studies were used asreported.

FIGS. 15A-15C show a comparison of tumor:tissue ratios at 4 h and 24 hto previous studies: (FIG. 15A) PC3 at 4 h; (FIG. 15B) PC3 at 24 h;(FIG. 15C) LNCaP at 4 and 24 h.

FIG. 16 shows tumoral uptake at 4 and 24 h post-injection in 4T1tumor-bearing female BALB/c mice with the various isotopes (¹⁸F, ⁶⁴Cu,^(67/68)Ga) with the NO2A chelator conjugated to two different ends ofthe peptide through a cysteine conjugation (C²Var3 and C²⁷Var3).

FIG. 17 shows biodistribution (% ID/g) of NO2A-C²⁷Var3 labeled compoundsin 4T1 tumor-bearing female BALB/c mice at 4, 6, and 24 h post-injectionwith significant tumoral uptake by the tumor of the ⁶⁷Ga labeledmaterial at 4 and 24 h.

FIG. 18 shows SPECT/CT imaging of ⁶⁷Ga-NO2A-cysVar3 in 4T1 tumor-bearingfemale BALB/c mice at 4 and 24 h p.i. with significant tumoral uptake.MIP is the maximum intensity projection. S, sagittal; C, coronal; and T,transverse, slices at the level of the tumor. The arrowheads indicatethe position of the tumor.

FIGS. 19A and 19B show PET (FIG. 19A) and SPECT/CT (FIG. 19B) imaging ofNO2A-C²⁷Var3 labeled compounds in 4T1 tumor-bearing female BALB/c miceat 4, 6, and 24 h post-injection. Arrowheads indicate where the tumor islocated within the mouse.

FIG. 20 shows PET (⁶⁸Ga, 2 and 4 h) images of the Ga-NO2A-cysVar3radioconjugates. The arrowheads point to the tumor. Significant kidneyuptake is observed in all images, but the tumor is visible at every timepoint with greatest tumor:background contrast at 24 h p.i. MIP, maximumintensity projection; S, sagittal; C, coronal; T, transverse.

FIG. 21 shows the general specific activities of exemplary compositionsof the present technology, where the notes are as follows:^(a)Approximated from the amount of total peptide added; ^(b)Calculatedfrom the UV/vis standard concentration curve; ^(c)In a separateexperiment, a specific activity of 1656 μCi/nmol was attained, but thelower specific activity material was used in the studies in order to becomparable throughout.

FIG. 22 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NOTA-WTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 23 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-WTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 24 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-WT (n=4 (3at 24 h)) administered via the lateral tail vein in female, BALB/c micewith orthotopic 4T1 breast cancer allografts implanted into the mammaryfat pad.

FIG. 25 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NOTA-Var3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 26 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-Var3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 27 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-Var3 (n=5(4 at 4 h)) administered via the lateral tail vein in female, BALB/cmice with orthotopic 4T1 breast cancer allografts implanted into themammary fat pad.

FIG. 28 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NOTA-Var7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 29 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-Var7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 30 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NOTA-Var7 (n=4)administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 31 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NOTA-WTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 32 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-WTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 33 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-WT(n=5) administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 34 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NOTA-Var3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 35 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-Var3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 36 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-Var3(n=5 (4 at 4 h)) administered via the lateral tail vein in female,BALB/c mice with orthotopic 4T1 breast cancer allografts implanted intothe mammary fat pad.

FIG. 37 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NOTA-Var7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 38 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-Var7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 39 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NOTA-Var7(n=5) administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 40 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysWTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 41 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysWTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 42 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysWT(n=3-5) administered via the lateral tail vein in female, BALB/c micewith orthotopic 4T1 breast cancer allografts implanted into the mammaryfat pad full graph from text. Glucose was administered i.p. 30 min priorto tracer injection.

FIG. 43 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysVar7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 44 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 45 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar7(n=4-5) administered via the lateral tail vein in female, BALB/c micewith orthotopic 4T1 breast cancer allografts implanted into the mammaryfat pad full graph from text. Glucose was administered i.p. 30 min priorto tracer injection.

FIG. 46 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysWTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 47 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NO2A-cysWTadministered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 48 shows the tissue uptake (mean % ID/g±SD) of [¹⁸F]—AlF-NO2A-cysWT(n=4) administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad full graph from text. Glucose was administered i.p. 30 min prior totracer injection.

FIG. 49 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar7administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 50 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar7 administered via the lateral tail vein in female,BALB/c mice with orthotopic 4T1 breast cancer allografts implanted intothe mammary fat pad.

FIG. 51 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar7 (n=3-5) administered via the lateral tail vein infemale, BALB/c mice with orthotopic 4T1 breast cancer allograftsimplanted into the mammary fat pad full graph from text. Glucose wasadministered i.p. 30 min prior to tracer injection.

FIG. 52 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 53 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 54 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar3(n=3-8) administered via the lateral tail vein in female, BALB/c micewith orthotopic 4T1 breast cancer allografts implanted into the mammaryfat pad full graph from text. Glucose was administered i.p. 30 min priorto tracer injection.

FIG. 55 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar3administered via the lateral tail vein in female, BALB/c mice withorthotopic 4T1 breast cancer allografts implanted into the mammary fatpad.

FIG. 56 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 administered via the lateral tail vein in female,BALB/c mice with orthotopic 4T1 breast cancer allografts implanted intothe mammary fat pad.

FIG. 57 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 (n=4-10) administered via the lateral tail veinin female, BALB/c mice with orthotopic 4T1 breast cancer allograftsimplanted into the mammary fat pad full graph from text. Glucose wasadministered i.p. 30 min prior to tracer injection.

FIG. 58 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderPC3 (prostate cancer) xenografts.

FIG. 59 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderPC3 (prostate cancer) xenografts.

FIG. 60 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderLNCaP (prostate cancer) xenografts.

FIG. 61 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderLNCaP (prostate cancer) xenografts.

FIG. 62 shows the tissue uptake (mean % ID±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in female, C57Bl/6 mice withorthotopic B16-F10 (melanoma) allografts.

FIG. 63 shows the tissue uptake (mean % ID/g±SD) of ⁶⁴Cu-NO2A-cysVar3administered via the lateral tail vein in female, C57Bl/6 mice withorthotopic B16-F10 (melanoma) allografts.

FIG. 64 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderPC3 (prostate cancer) xenografts.

FIG. 65 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 administered via the lateral tail vein in male,nude mice with shoulder PC3 (prostate cancer) xenografts.

FIG. 66 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with shoulderLNCaP (prostate cancer) xenografts.

FIG. 67 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 administered via the lateral tail vein in male,nude mice with shoulder LNCaP (prostate cancer) xenografts.

FIG. 68 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar3administered via the lateral tail vein in female, C57Bl/6 mice withorthotopic B16-F10 (melanoma) allografts and one group of female, BALB/cmice with orthotopic B16-F10 (melanoma) allografts at 6 h in therightmost column and further indicated by the asterisk (*).

FIG. 69 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 administered via the lateral tail vein in female,C57Bl/6 mice with orthotopic B16-F10 (melanoma) allografts and one groupof female, BALB/c mice with orthotopic B16-F10 (melanoma) allografts at6 h in the rightmost column and further indicated by the asterisk (*).

FIG. 70 shows the tissue uptake (mean % ID±SD) of [¹⁸F]—AlF-NO2A-cysVar3administered via the lateral tail vein in male, nude mice with U87MGxenografted flank tumors at 6 h.

FIG. 71 shows the tissue uptake (mean % ID/g±SD) of[¹⁸F]—AlF-NO2A-cysVar3 administered via the lateral tail vein in male,nude mice with U87MG xenografted flank tumors at 6 h.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the present methods are described below invarious levels of detail in order to provide a substantial understandingof the present technology.

In practicing the present methods, many conventional techniques inmolecular biology, protein biochemistry, cell biology, microbiology andrecombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001)Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubelet al. eds. (2007) Current Protocols in Molecular Biology; the seriesMethods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al.(1991) PCR 1: A Practical Approach (IRL Press at Oxford UniversityPress); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow andLane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gaited. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames andHiggins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) NucleicAcid Hybridization; Hames and Higgins eds. (1984) Transcription andTranslation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal(1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring HarborLaboratory); Makrides ed. (2003) Gene Transfer and Expression inMammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods inCell and Molecular Biology (Academic Press, London); and Herzenberg etal. eds (1996) Weir's Handbook of Experimental Immunology.

The present disclosure provides compounds, complexes, and compositionsthat include modified pH (low) insertion peptides that exhibit hightumoral uptake, prolonged tumor retention, and minimal accumulation innon-target organs and tissues. These complexes and compositions areuseful as diagnostic imaging agents because they permit diagnosticimaging over a wider-range of times, rather than shorter time points(e.g., fludeoxyglucose F 18 (“¹⁸F-FDG”), a radiopharmaceutical used inthe medical imaging modality positron emission tomography) or muchlonger time points (e.g., radiolabeled antibodies) in order to detectacidic tissues, such as cancerous cells. The present disclosure providesa comparison of the pharmacokinetic properties of exemplary compounds ofthe present technology that chelate with different PET and SPECTradionuclides in various cancer models (e.g., an orthotopic breastcancer model (murine 4T1 mammary adenocarcinoma, a model oftriple-negative human stage IV breast cancer), melanoma, prostate, andbrain tumor models). Radiolabels that are incorporated into thecompositions of the present technology may have different half-lives:the SPECT isotope ⁶⁷Ga has a long half-life of 3.26 days, ⁶⁴Cu has amoderately short half-life of 12.7 hours, whereas the half-life of ¹⁸Fand ⁶⁸Ga are 109.8 min and 67.7 min respectively. Unlike conventionalnucleophilic ¹⁸F labeling of small organic molecules, using a chelatorto coordinate the aluminum center of [¹⁸F]—AlF can be done in aqueoussolution.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this technology belongs. As used inthis specification and the appended claims, the singular forms “a”, “an”and “the” include plural referents unless the content clearly dictatesotherwise. For example, reference to “a cell” includes a combination oftwo or more cells, and the like. Generally, the nomenclature used hereinand the laboratory procedures in cell culture, molecular genetics,organic chemistry, analytical chemistry, biochemistry and hybridizationdescribed below are those well-known and commonly employed in the art.

As used herein, the term “about” in reference to a number is generallytaken to include numbers that fall within a range of 1%, 5%, or 10% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (e.g., exceptwhere such number would be less than 0% or exceed 100% of a possiblevalue).

As used herein, the “administration” of an agent or drug to a subjectincludes any route of introducing or delivering to a subject a compoundto perform its intended function. Administration can be carried out byany suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, intralesionally, orsubcutaneously), rectally, topically, intraarterially, intrathecally, orvia inhalation or via introduction into the cerebrospinal fluid.Administration includes self-administration and the administration byanother.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogues thatfunction in a manner similar to the naturally-occurring amino acids.Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acids canbe referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. In anyembodiment herein, the peptides included in the compounds and complexesof the present technology may include only D-amino acids.

As used herein, the term “cancer” refers to a malignant neoplasm ortumor (Stedman's Medical Dictionary, 25th ed.; Hensly ed.; Williams &Wilkins: Philadelphia, 1990). Exemplary cancers include acousticneuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma(e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma);appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g.,cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast); brain cancer (e.g., meningioma,glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma),medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer(e.g., cervical adenocarcinoma); choriocarcinoma; chordoma;craniopharyngioma; connective tissue cancer; epithelial carcinoma;ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multipleidiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterinecancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of theesophagus, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g.,intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gallbladder cancer; gastric cancer (e.g., stomach adenocarcinoma);gastrointestinal stromal tumor (GIST); germ cell cancer; head and neckcancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g.,oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer,pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer));hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia(ALL) (e.g., B cell ALL, T cell ALL), acute myelocytic leukemia (AML)(e.g., B cell AML, T cell AML), chronic myelocytic leukemia (CML) (e.g.,B cell CML, T cell CML), and chronic lymphocytic leukemia (CLL) (e.g., Bcell CLL, T cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., Bcell HL, T cell HL) and non Hodgkin lymphoma (NEIL) (e.g., B cell NHLsuch as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B celllymphoma), follicular lymphoma, chronic lymphocytic leukemia/smalllymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginalzone B cell lymphomas (e.g., mucosa associated lymphoid tissue (MALT)lymphomas, nodal marginal zone B cell lymphoma, splenic marginal zone Bcell lymphoma), primary mediastinal B cell lymphoma, Burkitt lymphoma,lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia),hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursorB lymphoblastic lymphoma and primary central nervous system (CNS)lymphoma; and T cell NHL such as precursor T lymphoblasticlymphoma/leukemia, peripheral T cell lymphoma (PTCL) (e.g., cutaneous Tcell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome),angioimmunoblastic T cell lymphoma, extranodal natural killer T celllymphoma, enteropathy type T cell lymphoma, subcutaneous panniculitislike T cell lymphoma, and anaplastic large cell lymphoma); a mixture ofone or more leukemia/lymphoma as described above; and multiple myeloma(MM)), heavy chain disease (e.g., alpha chain disease, gamma chaindisease, mu chain disease); hemangioblastoma; hypopharynx cancer;inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidneycancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma);liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma);lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer(SCLC), non small cell lung cancer (NSCLC), adenocarcinoma of the lung);leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); musclecancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferativedisorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis(ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF),chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CIVIL),chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES);neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreaticneuroendocrine tumor (GEP NET), carcinoid tumor); osteosarcoma (e.g.,bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma;pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer(e.g., Paget's disease of the penis and scrotum); pinealoma; primitiveneuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplasticsyndromes; intraepithelial neoplasms; prostate cancer (e.g., prostateadenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer;skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g.,appendix cancer); soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous glandcarcinoma; small intestine cancer; sweat gland carcinoma; synovioma;testicular cancer (e.g., seminoma, testicular embryonal carcinoma);thyroid cancer (e.g., papillary carcinoma of the thyroid, papillarythyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer;vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.”

As used herein, “radiolabel” refers to a moiety comprising a radioactiveisotope of at least one element. Exemplary suitable radiolabels includebut are not limited to those described herein. In some embodiments, aradiolabel is one used in positron emission tomography (PET). In someembodiments, a radiolabel is one used in single-photon emission computedtomography (SPECT). In some embodiments, radioisotopes comprise ¹⁸F (inthe form of [¹⁸F]—AlF²⁺), ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺,⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.

As used herein, the term “sample” refers to clinical samples obtainedfrom a subject or isolated microorganisms. In certain embodiments, asample is obtained from a biological source (i.e., a “biologicalsample”), such as tissue, bodily fluid, or microorganisms collected froma subject. Sample sources include, but are not limited to, mucus,sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), wholeblood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum,or tissue.

As used herein, the terms “subject,” “individual,” or “patient” are usedinterchangeably and refer to an individual organism, a vertebrate, amammal, or a human. In certain embodiments, the individual, patient orsubject is a human.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup is substituted with one or more substituents, unless otherwisespecified. In some embodiments, a substituted group is substituted with1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groupsinclude: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy,aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy,and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters;urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols;sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e.,SF₅), sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and ring systemsin which a bond to a hydrogen atom is replaced with a bond to a carbonatom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

Alkyl groups include straight chain and branched chain alkyl groupshaving from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkylgroups may be substituted or unsubstituted. Examples of straight chainalkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branchedalkyl groups include, but are not limited to, isopropyl, iso-butyl,sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropylgroups. Representative substituted alkyl groups may be substituted oneor more times with substituents such as those listed above, and includewithout limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl,thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl,carboxyalkyl, and the like.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the presenttechnology are designated by use of the suffix, “ene.” For example,divalent alkyl groups are alkylene groups, divalent aryl groups arearylene groups, divalent heteroaryl groups are divalent heteroarylenegroups, and so forth. Substituted groups having a single point ofattachment to the compound of the present technology are not referred tousing the “ene” designation. Thus, e.g., chloroethyl is not referred toherein as chloroethylene.

The term “halogen” or “halo” as used herein refers to bromine, chlorine,fluorine, or iodine. In some embodiments, the halogen is fluorine. Inother embodiments, the halogen is chlorine or bromine.

The term “hydroxyl” as used herein can refer to —OH or its ionized form,—O⁻. A “hydroxyalkyl” group is a hydroxyl-substituted alkyl group, suchas HO—CH₂—.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 atoms refers to groupshaving 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers togroups having 1, 2, 3, 4, or 5 atoms, and so forth.

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the present technology and include acid or baseaddition salts which retain the desired pharmacological activity and isnot biologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When the compound ofthe present technology has a basic group, such as, for example, an aminogroup, pharmaceutically acceptable salts can be formed with inorganicacids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuricacid, and phosphoric acid), organic acids (e.g., alginate, formic acid,acetic acid, trifluoroacetic acid, benzoic acid, gluconic acid, fumaricacid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid,succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid,naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic aminoacids (such as aspartic acid and glutamic acid). When the compound ofthe present technology has an acidic group, such as for example, acarboxylic acid group, it can form salts with metals, such as alkali andalkaline earth metals (e.g., Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), ammonia ororganic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine,pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) orbasic amino acids (e.g., arginine, lysine and ornithine). Such salts canbe prepared in situ during isolation and purification of the compoundsor by separately reacting the purified compound in its free base or freeacid form with a suitable acid or base, respectively, and isolating thesalt thus formed.

Those of skill in the art will appreciate that compounds of the presenttechnology may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or stereoisomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, stereochemical orgeometric isomeric forms, it should be understood that the presenttechnology encompasses any tautomeric, conformational isomeric,stereochemical and/or geometric isomeric forms of the compounds havingone or more of the utilities described herein, as well as mixtures ofthese various different forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The presence and concentrations of theisomeric forms will depend on the environment the compound is found inand may be different depending upon, for example, whether the compoundis a solid or is in an organic or aqueous solution. For example, inaqueous solution, quinazolinones may exhibit the following isomericforms, which are referred to as tautomers of each other:

As another example, guanidines may exhibit the following isomeric formsin protic organic solution, also referred to as tautomers of each other:

Because of the limits of representing compounds by structural formulas,it is to be understood that all chemical formulas of the compoundsdescribed herein represent all tautomeric forms of compounds and arewithin the scope of the present technology.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these stereoisomersare all within the scope of the present technology.

The compounds of the present technology may exist as solvates,especially hydrates. Hydrates may form during manufacture of thecompounds or compositions comprising the compounds, or hydrates may formover time due to the hygroscopic nature of the compounds. Compounds ofthe present technology may exist as organic solvates as well, includingDMF, ether, and alcohol solvates among others. The identification andpreparation of any particular solvate is within the skill of theordinary artisan of synthetic organic or medicinal chemistry

Compositions of the Present Technology

pH (low) insertion peptides represent a unique class of delivery agentsthat can target acidic malignant tissue. Without being bound by theory,the molecular mechanism of targeting is based on the pH-dependentformation of a transmembrane alpha helix, which is accompanied by theinsertion of pH (low) insertion peptides into the cellular membrane inenvironments with relatively low extracellular pH. FIG. 1 shows threefolding states of pH (low) insertion peptides in decreasingextracellular pH: unfolded and unbound peptide in solution (State I),unfolded peptide loosely interacting with the membrane lipid bilayer atphysiological pH (State II), and folded peptide in alpha-helicalconformation inserted across the membrane at low extracellular pH (StateIII).

In an aspect, a compound or pharmaceutically acceptable salt thereof isprovided, where the compound includes a pH (low) insertion peptide(“pHLIP”) configured to localize to an extracellular environment havinga pH that is lower than 7.4, wherein the pHLIP comprises a C-terminusand an N-terminus; and X¹ covalently attached to a heteroatom of a sidechain of an amino acid residue of the pHLIP, where the amino acidresidue is from 0, 1, 2, 3, 4, 5, or 6 residues from the C-terminus orthe N-terminus. An amino acid residue that is 0 residues from theN-terminus will be understood to mean the amino acid residue is theN-terminal residue. Similarly, an amino acid residue that is 0 residuesfrom the C-terminus will be understood to mean the amino acid residue isthe C-terminal residue. The extracellular environment may have a pH thatis lower than 7.1. For example, the extracellular environment may have apH that is 7.1, 7.0, 6.9, 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2,5.0, 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6, 3.4, 3.2, 3.0, 2.8, 2.6, 2.4,2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, or any range including and/or inbetween any two of these values. A tissue may include the extracellularenvironment. Such tissues include, but are not limited to,atherosclerotic plaques, ischemic myocardium, tissues impacted bystroke, and cancer tissues (such as solid tumors). Exemplary cancertissues include, but are not limited to, breast cancer, colorectalcancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, hepatocellular carcinoma, brain tumors, lung cancer, gastricor stomach cancer, pancreatic cancer, thyroid cancer, kidney or renalcancer, prostate cancer, melanoma, sarcomas, carcinomas, Wilms tumor,endometrial cancer, glioblastoma, squamous cell cancer, astrocytomas,salivary gland carcinoma, vulvar cancer, penile carcinoma, andhead-and-neck cancer; the cancer tissue may be breast cancer, a braintumor, prostate cancer, melanoma, or a metastatic cancer thereof.

In the composition, X¹ is of Formula I

where Z¹ and Z² are each independently a lone pair of electrons (i.e.providing an oxygen anion) or H; and Z³ is —NH-alkylene-,—NH—CH₂CH₂—((poly(alkylene glycol))-,

where n is 1, 2, 3, 4, 5, 6, 7, or 8, or a bond. Z³ may be—NH—(CH₂)_(m)—, —NH—CH₂CH₂—(O—CH₂CH₂)_(p)—,

or a bond, where n is 1, 2, 3, 4, 5, 6, 7, or 8; m is 1, 2, 3, 4, 5, 6,7, or 8; andp is 1, 2, 3, 4, 5, 6, 7, or 8. In any embodiment herein, X¹may be of Formula Ia

where Z¹ and Z² are each independently a lone pair of electrons (i.e.providing an oxygen anion) or H.

The pHLIP may be any one described in U.S. Pat. Nos. 8,076,451,8,703,909, 8,846,081, 9,676,823, and 9,289,508, each of which areincorporated herein by reference for any and all purposes, as well asU.S. Patent Application Nos. 2015/0086617 and 2016/0256560, each ofwhich are incorporated herein by reference for any and all purposes. Theamino acid residue of the pHLIP (to which heteroatom of the side chainX¹ is covalently attached) may be a cysteine or lysine. In anyembodiment herein, the X¹ may be covalently attached to a sulfur atom ofa cysteine residue of the pHLIP or is covalently attached to aε-nitrogen atom of a lysine residue the pHLIP. For sake a clarity, arepresentation of the amino acid lysine is provided below indicating theε-nitrogen atom.

In any embodiment herein, the pHLIP may be

(SEQ ID NO: 1) ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT, (SEQ ID NO: 2)ACDDQNPWRAYLDLLFPTDTLLLDLLW, (SEQ ID NO: 3)ADDQNPWRAYLDLLFPTDTLLLDLLWCG, or (SEQ ID NO: 4)ACEEQNPWARYLEWLFPTETLLLEL.

In any embodiment herein, the composition of the present technology maybe AC(X¹)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO:5),AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7),AC(X¹)EEQNPWARYLEWLFPTETLLLEL (SEQ ID NO:8), or a pharmaceuticallyacceptable salt of any one of these. For example, the composition may beAC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6), orADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7) where X¹ is of FormulaIa.

In a related aspect, a complex is provided that includes any embodimentof the composition described herein and a radionuclide. In such acomplex, X¹ may be of Formula II

where Z¹ and Z² are each independently a lone pair of electrons (i.e.providing an oxygen anion) or H; Z³ is —NH-alkylene-,—NH—CH₂CH₂—((poly(alkylene glycol))-,

where n is 1, 2, 3, 4, 5, 6, 7, or 8, or a bond; and M¹ is ⁶⁰Cu²⁺,⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or⁷¹Ga³⁺. In any embodiment herein, X¹ may be of Formula IIa

where Z¹ and Z² are each independently a lone pair of electrons (i.e.providing an oxygen anion) or H; and M¹ is ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺,⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺. Forexample, the complex may be AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ IDNO:6), or ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7) where X¹ is ofFormula IIa and M¹ is AlF²⁺.

In another related aspect of the present technology, a composition isprovided that includes any one of the aspects and embodiments ofcompounds and/or complexes and a pharmaceutically acceptable carrier. Asused herein, a “pharmaceutically acceptable carrier” includes carriersand/or excipients. In a further related aspect, a pharmaceuticalcomposition is provided, the pharmaceutical composition including aneffective amount of any one of the embodiments of the complexesdescribed herein for imaging a tissue comprising an extracellularenvironment having a pH that is lower than 7.4 (or any value or rangedisclosed herein) and a pharmaceutically acceptable carrier. The tissuemay be an atherosclerotic plaque, an ischemic myocardium, a tissueimpacted by stroke, and/or a cancer tissue (such as a solid tumor).Exemplary cancer tissues (and exemplary solid tumors of such tissues)include, but are not limited to, breast cancer, colorectal cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,hepatocellular carcinoma, brain tumors, lung cancer, gastric or stomachcancer, pancreatic cancer, thyroid cancer, kidney or renal cancer,prostate cancer, melanoma, sarcomas, carcinomas, Wilms tumor,endometrial cancer, glioblastoma, squamous cell cancer, astrocytomas,salivary gland carcinoma, vulvar cancer, penile carcinoma, andhead-and-neck cancer; the cancer tissue may be breast cancer, a braintumor, prostate cancer, melanoma, or a metastatic cancer thereof. Suchcompositions and pharmaceutical compositions may be used in any methoddescribed herein.

“Effective amount” refers to the amount of a complex required to producea desired effect, such as a quantity of a complex of the presenttechnology necessary to be detected by the detection method chosen. Forexample, an effective amount of a complex of the present technologyincludes an amount sufficient to enable detection of binding of thecomplex to a target of interest including, but not limited to, one ormore of an atherosclerotic plaque, an ischemic myocardium, a tissueimpacted by stroke, and/or a cancer tissue (such as a solid tumor).Another example of an effective amount includes amounts or dosages thatare capable of providing a detectable positron emission and/or gamma rayemission from positron emission and annihilation (above background) in asubject with a tissue comprising an extracellular environment having apH that is lower than 7.4, such as, for example, statisticallysignificant emission above background. As used herein, a “subject” or“patient” is a mammal, such as a cat, dog, rodent or primate. Typicallythe subject is a human, and, preferably, a human suffering from orsuspected of suffering from a condition that includes a tissue includingan extracellular environment having a pH that is lower than 7.4 asdescribed herein. The term “subject” and “patient” may be usedinterchangeably.

The instant present technology provides pharmaceutical compositions andmedicaments comprising any of the compounds or complexes disclosedherein and a pharmaceutically acceptable carrier or one or moreexcipients or fillers (collectively, such carriers, excipients, fillers,etc., will be referred to as “pharmaceutically acceptable carriers”unless a more specific term is used). The compositions may be used inthe methods and imagings described herein. Such compositions andmedicaments include an effective amount of any complex as describedherein for imaging one or more of the herein-described tissues. Thepharmaceutical composition may be packaged in unit dosage form. Forexample, the unit dosage form is effective in imaging a tissue includingan extracellular environment having a pH that is lower than 7.4 whenadministered to a subject.

The pharmaceutical compositions and medicaments may be prepared bymixing one or more compounds or complexes of the present technology,pharmaceutically acceptable salts thereof, stereoisomers thereof,tautomers thereof, or solvates thereof, with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like to image disordersassociated with a tissue that includes an extracellular environmenthaving a pH that is lower than 7.4 when administered to a subject. Thecompounds and complexes described herein may be used to prepareformulations and medicaments for imaging a variety of disordersassociated with a tissue comprising an extracellular environment havinga pH that is lower than 7.4. Such compositions can be in the form of,for example, granules, powders, tablets, capsules, syrup, suppositories,injections, emulsions, elixirs, suspensions or solutions. The instantcompositions can be formulated for various routes of administration, forexample, by oral, parenteral, topical, rectal, nasal, vaginaladministration, or via implanted reservoir. Parenteral or systemicadministration includes, but is not limited to, subcutaneous,intravenous, intraperitoneal, and intramuscular, injections. Thefollowing dosage forms are given by way of example and should not beconstrued as limiting the instant present technology.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds or complexes of the instant present technology, orpharmaceutically acceptable salts or tautomers thereof, with at leastone additive such as a starch or other additive. Suitable additives aresucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch,agar, alginates, chitins, chitosans, pectins, tragacanth gum, gumarabic, gelatins, collagens, casein, albumin, synthetic orsemi-synthetic polymers or glycerides. Optionally, oral dosage forms cancontain other ingredients to aid in administration, such as an inactivediluent, or lubricants such as magnesium stearate, or preservatives suchas paraben or sorbic acid, or anti-oxidants such as ascorbic acid,tocopherol or cysteine, a disintegrating agent, binders, thickeners,buffers, sweeteners, flavoring agents or perfuming agents. Tablets andpills may be further treated with suitable coating materials known inthe art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution. An isotonicsolution will be understood as isotonic with the subject. Alternatively,sterile oils may be employed as solvents or suspending agents.Typically, the oil or fatty acid is non-volatile, including natural orsynthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Complexes of the present technology may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. The carriers and stabilizers varywith the requirements of the particular complex, but typically includenonionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant)aerosols are typically used for delivery of complexes of the presenttechnology by inhalation.

Dosage forms for the topical (including buccal and sublingual) ortransdermal administration of complexes of the present technologyinclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, and patches. The active component may be mixed under sterileconditions with a pharmaceutically-acceptable carrier or excipient, andwith any preservatives, or buffers, which may be required. Powders andsprays can be prepared, for example, with excipients such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. The ointments, pastes, creamsand gels may also contain excipients such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Absorption enhancers can also be used toincrease the flux of the compounds and complexes of the presenttechnology across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane (e.g., as part of atransdermal patch) or dispersing the compound and/or complex in apolymer matrix or gel.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instant presenttechnology. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

The formulations of the present technology may be designed to beshort-acting, fast-releasing, long-acting, and sustained-releasing asdescribed below. Thus, the pharmaceutical formulations may also beformulated for controlled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant present technology.

Those skilled in the art are readily able to determine an effectiveamount by simply administering a complex of the present technology to apatient in increasing amounts until, for example, statisticallysignificant resolution (via, e.g., positron emission tomography) of atissue comprising an extracellular environment having a pH that is lowerthan 7.4 is achieved. The complexes of the present technology may beadministered to a patient at dosage levels in the range of about 0.1 toabout 1,000 mg per day. For a normal human adult having a body weight ofabout 70 kg, a dosage in the range of about 0.01 to about 100 mg per kgof body weight per day is sufficient. The specific dosage used, however,can vary or may be adjusted as considered appropriate by those ofordinary skill in the art. For example, the dosage can depend on anumber of factors including the requirements of the patient, theseverity of the condition being imaged, and the pharmacological activityof the complex being used. The determination of optimum dosages for aparticular patient is well known to those skilled in the art. Variousassays and model systems can be readily employed to determine theeffectiveness of a complex according to the present technology.

The complexes of the present technology can also be administered to apatient along with other conventional imaging agents that may be usefulin the imaging of a tissue that includes an extracellular environmenthaving a pH that is lower than 7.4. Such tissues include, but are notlimited to, an atherosclerotic plaque, an ischemic myocardium, a tissueimpacted by stroke, and/or a cancer tissue (such as a solid tumor).Exemplary cancer tissues (and exemplary solid tumors of such tissues)include, but are not limited to, breast cancer, colorectal cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,hepatocellular carcinoma, brain tumors, lung cancer, gastric or stomachcancer, pancreatic cancer, thyroid cancer, kidney or renal cancer,prostate cancer, melanoma, sarcomas, carcinomas, Wilms tumor,endometrial cancer, glioblastoma, squamous cell cancer, astrocytomas,salivary gland carcinoma, vulvar cancer, penile carcinoma, andhead-and-neck cancer; the cancer tissue may be breast cancer, a braintumor, prostate cancer, melanoma, or a metastatic cancer thereof. Thus,a pharmaceutical composition of the present technology may furtherinclude an imaging agent different than the complex of the presenttechnology. The administration may include oral administration,parenteral administration, or nasal administration. In any of theseembodiments, the administration may be intravenously, intramuscularly,intraarterially, intrathecally, intracapsularly, intradermally,intraperitoneally, intralesionally, subcutaneously,intracerebroventricularly, orally, intranasally, rectally, topically, orvia inhalation. The methods of the present technology may also includeadministering, either sequentially or in combination with one or morecomplexes of the present technology, a conventional imaging agent in anamount that can potentially or synergistically be effective for theimaging of a tissue comprising an extracellular environment having a pHthat is lower than 7.4.

In an aspect, a complex of the present technology is administered to apatient in an amount or dosage suitable for imaging. Generally, a unitdosage comprising a complex of the present technology will varydepending on patient considerations. Such considerations include, forexample, age, protocol, condition, sex, extent of disease,contraindications, concomitant therapies and the like. An exemplary unitdosage based on these considerations can also be adjusted or modified bya physician skilled in the art. For example, a unit dosage for a patientcomprising a complex of the present technology can vary from 1×10⁻⁴ g/kgto 1 g/kg, preferably, 1×10⁻³ g/kg to 1.0 g/kg. Dosage of a complex ofthe present technology can also vary from 0.01 mg/kg to 100 mg/kg or,preferably, from 0.1 mg/kg to 10 mg/kg.

The terms “associated” and/or “binding” can mean a chemical or physicalinteraction, for example, between a compound or complex of the presenttechnology and a target of interest. Examples of associations orinteractions include covalent bonds, ionic bonds,hydrophilic-hydrophilic interactions, hydrophobic-hydrophobicinteractions and complexes. Associated can also refer generally to“binding” or “affinity” as each can be used to describe various chemicalor physical interactions. Measuring binding or affinity is also routineto those skilled in the art. For example, complexes of the presenttechnology can bind to or interact with a target of interest orprecursors, portions, fragments and peptides thereof and/or theirdeposits.

Diagnostic Methods of the Present Technology

In one aspect, the present disclosure provides a method for detectingsolid tumors in a subject in need thereof comprising (a) administeringto the subject an effective amount of a complex of any embodimentdescribed herein, wherein the complex is configured to localize to asolid tumor having an acidic pH environment (that is, an extracellularenvironment having a pH that is lower than 7.4); and (b) detecting thepresence of solid tumors in the subject by detecting radioactive levelsemitted by the complex that are higher than a reference value. In someembodiments, the subject is human. In some embodiments, the environmentof the solid tumor has a pH value that is 7.1, 7.0, 6.9, 6.8, 6.7, 6.6,6.5, 6.4, 6.3, 6.2, 6.1, 6.0 or lower. For example, the extracellularenvironment of the solid tumor may have a pH that is 7.1, 7.0, 6.9, 6.8,6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2, 5.0, 4.8, 4.6, 4.4, 4.2, 4.0,3.8, 3.6, 3.4, 3.2, 3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2,1.0, or any range including and/or in between any two of these values.

In some embodiments of the methods disclosed herein, the radioactivelevels emitted by the complex are detected using positron emissiontomography or single photon emission computed tomography.

Additionally or alternatively, in some embodiments of the methodsdisclosed herein, the subject is diagnosed with, is at risk for, or issuspected of having cancer. The cancer may be selected from the groupconsisting of breast cancer, colorectal cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, brain tumors, lung cancer, gastric or stomach cancer,pancreatic cancer, thyroid cancer, kidney or renal cancer, prostatecancer, melanoma, sarcomas, carcinomas, Wilms tumor, endometrial cancer,glioblastoma, squamous cell cancer, astrocytomas, salivary glandcarcinoma, vulvar cancer, penile carcinoma, and head-and-neck cancer;the cancer may be breast cancer, a brain tumor, prostate cancer,melanoma, or a metastatic cancer thereof.

Additionally or alternatively, in some embodiments of the methodsdisclosed herein, the complex is administered intravenously,intramuscularly, intraarterially, intrathecally, intracapsularly,intradermally, intraperitoneally, intralesionally, transtracheally,subcutaneously, intracerebroventricularly, orally, intranasally,rectally, topically, or via inhalation. In certain embodiments, thecomplex is administered into the cerebral spinal fluid or blood of thesubject. In some embodiments of the methods disclosed herein, theradioactive levels emitted by the complex are detected between 1 to 48hours after the complex is administered. For example, the radioactivelevels emitted by the complex may be detected at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, or 48 hours (or any subrange therein) after the complex isadministered.

In certain embodiments of the methods disclosed herein, the radioactivelevels emitted by the complex are expressed as the percentage injecteddose per gram tissue (% ID/g). The reference value may be calculated bymeasuring the radioactive levels present in non-tumor (normal) tissues,and computing the average radioactive levels present in non-tumor(normal) tissues±standard deviation. In some embodiments, the referencevalue is the standard uptake value (SUV). See Thie J A, J Nucl Med.45(9):1431-4 (2004). In some embodiments, the ratio of radioactivelevels between a tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1,50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

The effectiveness of such a complex may be determined by computing thearea under the curve (AUC) tumor: AUC normal tissue ratio. In someembodiments, the complex has an area under the curve (AUC) tumor: AUCnormal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1,70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

In another aspect, the present disclosure provides a method fordetecting acidic diseased tissue in a subject in need thereof comprising(a) administering to the subject an effective amount of a complex of anyembodiment described herein, wherein the complex is configured tolocalize to an extracellular environment having a pH that is lower than7.4; and (b) detecting the presence of acidic diseased tissue (that is,a tissue with an extracellular environment having a pH that is lowerthan 7.4) in the subject by detecting radioactive levels emitted by thecomplex that are higher than a reference value. In some embodiments, thesubject is human. Examples of acidic diseased tissue includeatherosclerotic plaques, ischemic myocardium, tissues impacted bystroke, and tumors. In some embodiments, the extracellular environmentof the acidic diseased tissue has a pH value that is 7.3, 7.2, 7.1, 7.0,6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0 or lower. For example,the extracellular environment of the tissue may have a pH that is 7.1,7.0, 6.9, 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2, 5.0, 4.8, 4.6,4.4, 4.2, 4.0, 3.8, 3.6, 3.4, 3.2, 3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8,1.6, 1.4, 1.2, 1.0, or any range including and/or in between any two ofthese values. The radioactive levels emitted by the complex areexpressed as the percentage injected dose per gram tissue (% ID/g).

In some embodiments of the methods disclosed herein, the radioactivelevels emitted by the complex are detected using positron emissiontomography or single photon emission computed tomography.

Additionally or alternatively, in some embodiments of the methodsdisclosed herein, the complex is administered intravenously,intramuscularly, intraarterially, intrathecally, intracapsularly,intraorbitally, intradermally, intraperitoneally, intralesionally,subcutaneously, intracerebroventricularly, orally, rectally, topically,vaginally, or via inhalation. In some embodiments of the methodsdisclosed herein, the radioactive levels emitted by the complex aredetected between 1 to 48 hours after the complex is administered. Forexample, the radioactive levels emitted by the complex may be detectedat 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours (or any subrangetherein) after the complex is administered.

Kits

The present technology provides kits containing components suitable fordiagnosing in a patient a tissue that includes an extracellularenvironment having a pH that is lower than 7.4, such as anatherosclerotic plaque, an ischemic myocardium, a tissue impacted bystroke, and/or a cancer tissue (such as a solid tumor). In one aspect,the kits include at least one compound or complex of the presenttechnology disclosed herein, instructions for use, and optionally atleast one radionuclide. The compound or complex may be provided in theform of a prefilled syringe or autoinjection pen containing a sterile,liquid formulation or lyophilized preparation of the compound or complex(e.g., Kivitz et al., Clin. Ther. 28:1619-29 (2006)).

Additionally or alternatively, in some embodiments of the kits of thepresent technology, the at least one radionuclide includes is selectedfrom among ⁶⁰Cu²⁺, ⁶¹CU²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺, ⁶⁷Ga³⁺,⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.

If the kit components are not formulated for oral administration, adevice capable of delivering the kit components through some other routemay be included. Examples of such devices include syringes (forparenteral administration) or inhalation devices. The kit components maybe packaged together or separated into two or more containers. In someembodiments, the containers may be vials that contain sterile,lyophilized formulations of a compound or complex of the presenttechnology that are suitable for reconstitution. A kit may also containone or more buffers suitable for reconstitution and/or dilution of otherreagents. Other containers that may be used include, but are not limitedto, a pouch, tray, box, tube, or the like. Kit components may bepackaged and maintained sterilely within the containers.

EXAMPLES Example 1: Materials and Methods for Generating the Compoundsand Complexes of the Present Technology

General. Synthesis of the D-amino acid versions of severalNOTA-derivatized pHLIPs were provided by CS Bio (Menlo Park, Calif.)under instruction of the inventors with ≥95% purity (combination of bothchelators (FIGS. 2A and 2B) with NO2A attached to two positions on Var3(FIGS. 2B and 2C) and peptide sequences to form the complexes in Table1: NOTA-WT, NOTA-Var3, NOTA-Var7, NO2A-cysWT, NO2A-cysVar3,NO2A-cysVar7, and NO2A-C²⁷Var3). In general, the NOTA-derivatized pHLIPswere synthesized via solid-phase synthesis of the peptide, subsequentremoval of the thiol-protecting group, addition of the NO2A-NHS ester(Maleimido-mono-amide-NOTA, B-622, Macrocyclics, Plano, Tex., USA),followed by cleavage from the resin before purification and analysis. Inthe instance of the NOTA-pHLIPs, the peptide was synthesized viasolid-phase peptide synthesis and the p-SCN-Bn-NOTA (B-605,Macrocyclics, Plano, Tex., USA) was added to react with the N-terminusof the peptide prior to cleaving from the resin. All other chemicalswere purchased from commercial suppliers without further purificationunless otherwise stated.

Table 1 shows pHLIP sequences from N-terminus and C-terminus withabbreviated name used in FIGS. 2A and 2B. NO2A-cysVar3 or NO2A-C²Var3 isthe NO2A-Var3 with the NO2A attached to a cysteine on the N-terminal(extracellular) portion of the peptide. The superscripted 2 representsthe cysteine's residue number in the particular pHLIPsequence.NO2A-C²⁷Var3 is the NO2A-Var3 with the NO2A attached to a cysteine onthe C-terminal (intracellular) portion of the peptide. The superscripted27 represents the cysteine's residue number in the particular pHLIPsequence.

TABLE 1 Name(s) Peptide Sequence* NOTA-WT(NOTA)-ACEQNPIYWARYADWLFTTPLLLLDLA LLVDADEGT (SEQ ID NO: 9) NOTA-Var3(NOTA)-ACDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: 10) NOTA-Var7(NOTA)-ACEEQNPWARYLEWLFPTETLLLEL (SEQ ID NO: 11) NO2A-cysWTAC(NO2A)EQNPIYWARYADWLFTTPLLLLDLAL (NO2A-C²WT) LVDADEGT (SEQ ID NO: 12)NO2A-cysVar3** AC(NO2A)DDQNPWRAYLDLLFPTDTLLLDLLW (NO2A-C²Var3)(SEQ ID NO: 13) NO2A-C²⁷Var3 ADDQNPWRAYLDLLFPTDTLLLDLLWC(NO2A)G(SEQ ID NO: 14) NO2A-cysVar7** AC(NO2A)EEQNPWARYLEWLFPTETLLLEL(NO2A-C²Var7) (SEQ ID NO: 15) *NO2A is listed in Macrocyclic's catalog(B-622) as the name: Maleimido-mono-amide-NOTA. NO2A is also known as1,4,7-Triazacyclononane-1,4-bis-acetic acid-7-maleimidoethylacetamide.**In prior literature, Var3 was referred to as Short3D and Var7 wasreferred to as Short3E.

Labeling Methods for ⁶⁴Cu-NOTA-pHLIP.

Following a preparation similar to Zeglis et al., J. Nuc. Med.54(8):1389-1396 (2013), 3.13-11.26 mCi (116-417 MBq) of ⁶⁴Cu in 0.1 MHCl (2.5-6.0 μL, 1.86×10⁴-1.25×10⁴ mCi/μmol, Washington University, St.Louis, Mo.) was added to 150 μL of 100 mM NH₄OAc buffer (pH ˜5.5). Analiquot (25-37 μL) of NOTA-pHLIP derivative (0.46-0.63 mM) in DMSO wasadded to the pH adjusted ⁶⁴Cu solution. The solution was placed on athermomixer (Eppendorf, Hamberg, Germany) at 80° C. for 15 min at 1100rpm. An aliquot of the crude product mixture was HPLC-analyzed (5-95%acetonitrile in water (with 0.1% trifluoroacetic acid) over 15 min,Jupiter C-18 column, Phenomenex, 250 mm×4.6 mm, 5 μm, 300 Å) to ensurethat product was present (⁶⁴Cu-NOTA-pHLIP eluted at 10.3 min;⁶⁴Cu-NO2A-cyspHLIP eluted at 13.1 min). The remainder of the reactionmixture was separated using a pretreated Oasis HLB Plus Light or C18Sep-Pak Light cartridge (Waters, Milford, Mass.). Free and unbound ⁶⁴Cuwas removed by washing the cartridge with 5 mL of water. The pure⁶⁴Cu-labeled NOTA-pHLIP derivatives were then eluted with 1 mL ethanolin 0.1 mL fractions. The ethanolic fractions with the highest activitieswere combined and diluted with sterile phosphate buffered saline (PBS)to provide <10% ethanol in the solution before HPLC analysis (JupiterC-18, 5 μm, 300 Å, 250×4.6 mm, Phenomenex; 5-95% AcN in water with 0.1%TFA over 15 min at 1 mL/min) to ensure that no free ⁶⁴Cu was present.The radiochemical purity of the species was always >93% and the specificactivity (using the original amount of compound added to the reactionmixture) was estimated to be 56.0-318.4 μCi/nmol (3.05-11.8 GBq/μmol).The reaction yielded 1.29-9.54 mCi (47.7-353 MBq) of product with anisolated yield of 40.7-84.7%.

Alternatively, ⁶⁴Cu was diluted in a 100 mM NH₄OAc buffer (pH ˜5.5) andreacted with a NOTA-pHLIP derivative at 80° C. for 15 min.

Labeling Methods for [¹⁸F]—AlF-NOTA-pHLIP.

Following a modified labeling method described by Dijkgraaf et al., J.Nuc. Med. 53(6):947-952 (2012), 52.0-60.8 mCi (1.92-2.25 GBq) of ¹⁸Ftarget water (produced from ¹⁸O enriched target water using a GEMSPETtrace-800 cyclotron, Memorial Sloan Kettering Cancer Center, NewYork, N.Y.) was obtained. The target water was loaded onto apreconditioned chromafix cartridge (30-PS—HCO₃, Advanced BiochemicalCompounds (ABX), Germany). All of the preconditioning solvents and theelution solution were prepared with metal-free water and trace-metalfree reagents. The ¹⁸F was eluted from the cartridge with approximately100 of 0.4 M KHCO₃ in metal-free water into a 1.5 mL tube (ThermoFisherScientific, Waltham, Mass.). The pH was adjusted to ˜4.1 with metal-freeacetic acid (Sigma Aldrich, St. Louis, Mo.). To the pH adjustedsolution, 100 μL of acetonitrile was added. If the total activityexceeded 50 mCi (1.85 GBq), the mixture was split between multiple 1.5mL tubes prior to continuing (˜10 mCi/40 nmol reaction). To thealiquotted mixture, 20 μL (40 nmol) of 2 mM AlCl₃ in 0.1 M NH₄OAc buffer(pH ˜4.1) was added to each tube. The solution was allowed to react for5 min at room temperature before 14-17 μL of 4-5 mM NOTA-pHLIP in DMSO(68-75 nmol) was added, the tube capped, and the reaction mixturereacted at 75° C. for 15 minutes in a thermomixer. After reaction, themixture was diluted with 2 mL of metal-free water. An aliquot of thereaction mixture was HPLC-analyzed (20-95% acetonitrile in water (with0.1% trifluoroacetic acid) over 15 min with an Atlantis T3, Waters, 250mm×4.6 mm, 5 μm; Jupiter, Phenomenex, 250 mm×4.6 mm, 5 μm, 300 Å,column) to ensure that product was formed ([¹⁸F]—AlF-NOTA-WT elutedbetween 10.1 and 12.1 min; [¹⁸F]—AlF-NOTA-var3 eluted between 10.2 and11.4 min; [¹⁸F]—AlF-NOTA-var7 eluted between 8.2 and 10.3 min;[¹⁸F]—AlF-NOTA-cysWT eluted between 13 and 16 min;[¹⁸F]—AlF-NOTA-cysVar3 eluted between 12 and 15 min;[¹⁸F]—AlF-NOTA-cysVar7 eluted between 12 and 14 min). While the HPLCanalysis was being performed, the remainder of the reaction mixture wasseparated using a pretreated Oasis HLB Plus Light or C18 Sep-Pak Lightcartridge (Waters, Milford, Mass.) eluting first with water (5 mL) toremove any unbound ¹⁸F species and then ethanol (0.1 mL fractions for 4fractions and then 0.6 mL). The [¹⁸F]—AlF-NOTA-pHLIP derivatives elutedwithin the first three fractions in ethanol. The ethanolic solution wasdiluted with sterile PBS to provide <10% ethanol in the solution beforeHPLC analysis (Jupiter C-18, 5 μm, 300 Å, 250×4.6 mm, Phenomenex; 5-95%AcN in water with 0.1% TFA over 15 min at 1 mL/min). The radiochemicalpurity of the species was always ≥95% and the specific activity (usingthe original amount of compound added to the reaction mixture) wasestimated to be 79.9-178.1 μCi/μmol (2.96-6.60 GBq/μmol). The reactionyielded 6.02-9.24 mCi (223-342 MBq) with an overall, decay-corrected (tobeginning of labeling procedure) isolated yield of 2.33-48.6%.

Alternatively, ¹⁸F from the cyclotron target water was concentratedusing a chromafix cartridge into 0.1 mL of 0.4 M KHCO₃ in metal-freewater. The pH was adjusted and 0.1 mL AcN added. To this solution, AlCl₃(80 nmol) in 0.1 M NH₄OAc buffer (pH 4.1) was added. After 5 min, theNOTA-pHLIP derivative was added and the reaction mixture reacted at 75°C. for 15 min.

General Labeling Methods for Cu-NOTA-pHLIP.

Approximately 200 μL of 36.4 M Cu(OAc)₂ in 0.1 M NH₄OAc (pH ˜5) inmetal-free water was added to 5-6 mg of NOTA-pHLIP in approximately 200μL of DMSO. The mixtures were reacted in a thermomixer at 50° C. for 1h, and then filtered through a 0.2 μm filter. The filter was washed withan additional 1 mL of 50% AcN in water and 2 mL of water, sequentially.The combined filtrate was HPLC purified (10-95% AcN in water over 30 minwith a Jupiter C-18 column, 5 μm, 300 Å, 250×4.6 mm, Phenomenex). Thecollected fraction (14.5-15.5 min for Cu-NOTA-WT, 13.9-15 min forCu-NOTA-Var3, 13.2-14.1 min for Cu-NOTA-Var7, 12.2-13.5 min forCu-NO2A-cysWT, 13-15.3 min for Cu-NO2A-cysVar3, and 12.5-16 minCu-NO2A-cysVar7) was lyophilized.

General Labeling Methods for AlF-NOTA-pHLIP:

Sodium fluoride (19.8 mg, 0.47 mmol) was dissolved in 150 μL of 0.1 MNH₄OAc in metal-free water (pH ˜4.1). The resulting solution was addedto 13.7 mg AlCl₃ (0.057 mmol) with 50 μL of 0.1 M NH₄OAc buffer (pH˜4.1). The AlF₃ solution (60 μL) was added to approximately 7 mg ofNOTA-pHLIP dissolved in 60 μL of EtOH. The reaction mixtures werereacted at 50° C. on a thermomixer for 1 h before being HPLC purified(10-95% AcN in water over 30 min with a Jupiter column, 5 μm, 300 Å,250×4.6 mm, Phenomenex). The collected fraction (14.7-16.5 min forAIF-NOTA-WT, 13.8-15.4 min for AIF-NOTA-Var3, 13-14.6 min forAlF-NOTA-Var7, 12.5-17.5 min for AIF-NO2A-cysWT and AIF-NO2A-cysVar3,and 12-16 min AIF-NO2A-cysVar7) was lyophilized.

General Labeling Methods for ⁶⁷Ga-NOTA-pHLIP.

⁶⁷Ga-citrate was obtained from Nuclear Diagnostic Products (Rockaway,N.J.), trapped on a silica cartridge, washed with metal-free water, andeluted from the cartridge in 0.2 mL of 0.4 M HCl. The pH was adjusted toapproximately pH 4 with 1.0 M Na₂CO₃ and metal-free acetic acid. Thereaction was diluted with 0.2 mL AcN and a DMSO solution of NOTA-pHLIPadded. The reaction mixture was heated at 75-80° C. for 15 min. Thereaction mixture was diluted with water, loaded on a pretreated C18cartridge (Waters, Milford, Mass.), eluted with water (to removeunlabeled Ga), and the desired product eluted with EtOH. The ethanolicsolution was diluted to <10% with sterile PBS for animal injections andHPLC analysis (all >95% R.C.P.). The biodistribution of theradioconjugates were evaluated at 1, 2, 4, and 24 h p.i. (⁶⁷Ga) in 4T1orthotopic tumor-bearing BALB/c female mice. PET/CT (2 and 4 h p.i.) andSPECT/CT (6 and 24 h p.i.) imaging studies were carried out.

General Labeling Methods for ⁶⁸Ga-NOTA-pHLIP.

⁶⁸Ga was eluted from a ⁶⁸Ge/⁶⁸Ga generator in approximately 0.5 mL of0.5 M KOH. The pH of the eluant was adjusted to approximately 4 withmetal-free acetic acid. The NOTA-pHLIP was added in a DMSO solution andthe reaction reacted at 80° C. for 15 min. The reaction mixture wasdiluted with water, loaded on a pretreated C18 cartridge (Waters,Milford, Mass.), eluted with water (to remove unlabeled Ga), and thedesired product eluted with EtOH. The ethanolic solution was diluted to<10% with sterile PBS for animal injections and HPLC analysis (all >95%R.C.P.). The biodistribution of the radioconjugates were evaluated at 2and 4 h p.i. (⁶⁸Ga) in 4T1 orthotopic tumor-bearing BALB/c female mice.PET/CT (2 and 4 h p.i.) and SPECT/CT (6 and 24 h p.i.) imaging studieswere carried out.

4T1 Cell Culture.

The 4T1 cells derived from spontaneous breast tumor in a BALB/c mouse(Karmanos Cancer Institute, Detroit, Mich.) were cultured in Dulbecco'smodified Eagle's high glucose media with 10% FCS, 2 mM L-glutamine,penicillin, and streptomycin. The 4T1 cells derived from ATCC (Manassas,Va.) were cultured in RPMI-1640 medium modified to contain 2 mML-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/LNaHCO₃, penicillin, and streptomycin.

4T1 Orthotopic BALB/c Mouse Model.

The cells were removed from the flasks, concentrated, and resuspended inminimal media for cell counting. The cells were then diluted to containapproximately 1 million cells in 30 μL of media (for orthotopicallografts). Following an MSKCC Institutional Animal Care and UseCommittee approved protocol, 8-10 week old BALB/c mice (Charles RiverLaboratories, Wilmington, Mass.) were surgically implanted with onemillion 4T1 cancer cells into the first or the last mammary fat pad ofthe right side of the animal using aseptic surgical techniques andsterile staple closures while the mice were under anesthetic.Additionally, mice were given injections of meloxicam (24 h pain killer)in the scruff and bupivacaine intradermally prior to surgical incisions.One day post surgery, the mice were again given meloxicam and checked toensure that the closure was healing. Two and three days post surgery,the mice were checked to ensure that the animals were healthy andrecovering well from the surgery. Five to seven days post surgery, thestaples were removed. Five to nine days post surgery, the mice wereinjected with 50-75 μCi (0.9-0.4 nmol) of the respectiveradiopharmaceutical for in vivo biodistribution or 500-600 μCi (9-11nmol) for in vivo imaging. The mice receiving ⁶⁴Cu-NOTA-pHLIP weresacrificed at 1, 2, 4, 12, 24, 36, and 48 h; the mice receiving[′⁸F]—AlF-NOTA-pHLIP were sacrificed at 0.5, 1, 2, 4, 6 and 8 h. The 4T1tumors in this study were purposely used 5-9 days after inoculation toensure lower tumoral pH.

PC3 Cell Culture.

The prostate cancer PC3 cells, derived from a human bone metastasis,were purchased from ATCC (Manassas, Va.) and cultured in F-12K Medium(Kaighn's Modification of Ham's F-12 Medium) with, 10% FCS, 2 mML-glutamine, 1.5 g/L NaHCO₃, penicillin, and streptomycin.

PC3 Shoulder Nude Mouse Xenografts.

The cells were removed from the flasks, concentrated, and resuspended inminimal media for cell counting. The cells were then diluted to containapproximately 3 million cells in 150 μL of 1:1 media and matrigel (forshoulder xenografts). Following an MSKCC Institutional Animal Care andUse Committee approved protocol, 150 μL of the cell suspension wasinjected subcutaneously in 6-9 week old nude male mice (CRL). Mice wereused in studies at 3 weeks post inoculation. The mice were injected with50-75 μCi (0.9-0.4 nmol) of the respective radiopharmaceutical for invivo biodistribution or 500-600 μCi (9-11 nmol) for in vivo imaging. Themice receiving ⁶⁴Cu-NOTA-cysVar3 were sacrificed at 1, 4, 12, 24, and 48h; the mice receiving [¹⁸F]—AlF-NOTA-cysVar3 were sacrificed at 1, 4, 6and 8 h.

LNCaP Cell Culture.

The LNCaP cells, prostate cancer derived from a human supraclavicularlymph node metastasis, were purchased from ATCC (Manassas, Va.) andcultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mMHEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L NaHCO₃,penicillin, and streptomycin.

LNCaP Shoulder Nude Mouse Xenografts.

The cells were removed from the flasks, concentrated, and resuspended inminimal media for cell counting. The cells were then diluted to containapproximately 3-6 million cells in 150 μL of 1:1 media and matrigel (orshoulder xenografts). Following an MSKCC Institutional Animal Care andUse Committee approved protocol, 150 μL of the cell suspension wasinjected subcutaneously in 6-8 week old nude male mice (CRL). Mice wereused in studies at 3-8 weeks post inoculation. The mice were injectedwith 50-75 μCi (0.9-0.4 nmol) of the respective radiopharmaceutical forin vivo biodistribution or 500-600 μCi (9-11 nmol) for in vivo imaging.The mice receiving 64Cu-NOTA-cysVar3 were sacrificed at 1, 4, 12, 24,and 48 h; the mice receiving [¹⁸F]—AlF-NOTA-cysVar3 were sacrificed at1, 4, 6 and 8 h.

B16-F 10 Cell Culture.

The B16-F10 cells, derived from spontaneous melanoma in a C57Bl/6 mouse,were purchased from ATCC (Manassas, Va.) and cultured in Dubelco'smodified Eagle's high glucose media with 10% FCS, 4 mM L-glutamine, 1 mMsodium pyruvate, 1.5 g/L NaHCO₃, penicillin, and streptomycin.

Orthotopic B16-F 10 Shoulder Allografts.

The cells were removed from the flasks, concentrated, and resuspended inminimal media for cell counting. The cells were then diluted to containapproximately 100,000 cells in 100 μL of media (for shoulderallografts). Following an MSKCC Institutional Animal Care and UseCommittee approved protocol, C57Bl/6 female mice (CRL) and BALB/c femalemice (CRL) were injected with 100 μL of the cell suspensionintradermally as per a media substituted protocol from Overwijk &Restifo (Current Protocols in Immunology, 2000: 20.1.1-20.1.29). Themice were used in studies at approximately 9-14 days post inoculation.The mice were injected with 50-75 μCi (0.9-0.4 nmol) of the respectiveradiopharmaceutical for in vivo biodistribution or 500-600 μCi (9-11nmol) for in vivo imaging. The mice receiving 64Cu-NOTA-cysVar3 weresacrificed at 1, 4, 12, 14.5, and 24 h; the mice receiving[¹⁸F]—AlF-NOTA-cysVar3 were sacrificed at 1, 4, 6 and 8 h.

U-87 MG Cell Culture.

The human glioblastoma cell line, U-87 MG, was purchased from AmericanType Culture Collection (ATCC, Manassas, Va.) and cultured in Dulbecco'sModified Eagle's Medium with 10% fetal bovine serum, 2 mM L-Glutamine,1500 mg/L Sodium Bicarbonate, 100 units/mL penicillin G and 100 μg/mLstreptomycin (prepared by the MSKCC Media Preparation Core).

U-87 MG Shoulder Xenografts:

The cells were removed from the flasks, concentrated, and resuspended inminimal media for cell counting. The cells were then diluted to containapproximately 1 million cells in 150 μL of 1:1 media and matrigel (orshoulder xenografts). Following an MSKCC Institutional Animal Care andUse Committee approved protocol, 150 μL of the cell suspension wasinjected subcutaneously in 12 week old nude male mice (CRL). Mice wereused in studies at 2-3 weeks post inoculation. The mice were injectedwith 400-500 μCi (7-10 nmol) for in vivo imaging. The mice weresacrificed at 6 h after imaging (n=6).

General Purification.

An Oasis HLB Plus Light or C18 Sep-Pak Light cartridge (Waters, Milford,Mass.) was used to remove unbound activity. The pure radiolabeledNOTA-pHLIP derivatives were eluted with EtOH and diluted with sterilephosphate buffered saline (PBS) with purities >93% before administrationto animals.

Animal Models.

Animal studies were conducted according to MSKCC IACUC-approved animalprotocol. 4T1 (orthotopic breast cancer allografts of 1×10⁶ cellssurgically implanted in the mammary fat pad) and B16-F10 (orthotopicmelanoma allografts of 1×10⁵ cells injected intradermally on theshoulder) tumors were orthotopically inoculated in media in femaleBALB/c and C57Bl/6 mice (Charles River Laboratories (CRL)),respectively. Additionally, PC3 (3×10⁶ cells) or LNCaP (3-6×10⁶ cells)tumors were subcutaneously xenografted on the shoulder of athymic nudemale mice (CRL). Mice were used once tumor volumes were approximately90-300 mm³.

In Vivo Animal PET Imaging and Biodistribution.

Mice were injected with radiolabeled NOTA-pHLIP derivatives i.v.(500-700 μCi/mouse for PET or SPECT imaging studies and 25-100 μCi/mousefor biodistribution studies). All injections were less than 200 μL with<10% EtOH in sterile PBS. PET images were obtained with the mice underanesthesia in an Inveon PET-CT or microPET Focus 120 (Siemens) at 0.5-48h p.i. All images were analyzed using ASIPro VM (Concorde Microsystems).SPECT images were obtained with the mice under anesthesia in aNanoSPECT/CT Plus at 4-24 h p.i. All SPECT images were reconstructedusing HiSPECT and analyzed using InVivoScope. Dissections for ex vivobiodistribution were performed on mice after CO₂ asphyxiation orcervical dislocation while anesthetized at reported time points. Weightof the syringe prior to injection and after injection was used todetermine the mass of injectate. Activity of the syringe prior toinjection and after injection was used to determine the percent ofinjectate administered. The mass injected was corrected by the percentof radioactivity injected. Four to five aliquots (10 μL) were weighedand counted as internal standards for each study. All of the collectedorgans were counted using an automatic gamma counter (Wizard 3”,PerkinElmer, Waltham, Mass.). The total injected dose was found as themass injected dose×fraction radioactivity injected×internal standardaverage counts/g. The percent injected dose (% ID) was determined as thecounts for the tissue×100/total injected dose. The % ID/g was calculatedas the % ID/tissue weight. The average and standard deviation of the %ID and % ID/g was determined using normal methods (n-1) for each set ofmice.

Ex Vivo Autoradiography, Staining, and Microscopy.

Tumors were excised from the mice, embedded into Tissue-Plus® O.C.T.(Scigen, Gardena, Calif.), stored at −20° C. until sectioning, and cutin sequential 10 μm sections. Select sections were exposed toautoradiography film (Fujifilm, GE Healthcare) for 1-3 days and readusing a typhoon photographic film scanner (GE Healthcare). Additionally,sections were stained with H&E for gross tumor microscopy and thenscanned.

Biophysical Studies.

Biophysical studies were conducted on nonradioactive standards of theNOTA-pHLIP and NO2A-cyspHLIP compounds. All experiments were conductedwith a 5 μM to 1 mM complex to lipid ratio. State I (complex with noliposomes in solution) and State II (complex in the presence ofliposomes) were measured at pH 8; for State III (complex in presence ofliposomes), the pH of solution was dropped to pH 4 using 2 M HCl.Experiments were conducted in 10 mM phosphate buffer (Sigma Aldrich, St.Louis, Mo.) either without additional ions or in the presence ofphysiological levels of free magnesium and calcium (0.65 mM and 1.25 mM,respectively).

Liposome Preparation.

Large unilamellar vesicles were prepared by extrusion.1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; Avanti PolarLipids), dissolved in chloroform at a concentration of 12.5 mg/mL,de-solvated by rotary evaporation, and dried under high vacuum for 2 h.The phospholipid film was then rehydrated in 10 mM phosphate buffer (pH8.0 with or without ions), vortexed, and extruded 15× through a 50 nmpore.

Steady-State Fluorescence.

Tryptophan fluorescence spectra were measured using a PC1 ISSspectrofluorometer (Champaign, Ill.) with temperature control set to 25°C. Fluorescence spectra were recorded with excitation and emission slitsset to 1 mm, using an excitation wavelength of 295 nm, with excitationand emission polarizers set to 54.7° and 0°, respectively.

Steady-State Circular Dichroism.

Circular dichroism measurements were taken using a MOS-450 spectrometer(Bio-Logic SAS, Claix, France) with temperature control set to 25° C. inthe range of 190 to 260 nm with step of 1.0 nm.

pH Dependence.

The pH-dependent insertion of the complex population was investigatedusing the shift in position of the wavelength of maximum intensity ofthe tryptophan fluorescence spectra by varying pH conditions between 4and 8. The pH after addition of HCl was measured using an Orion PerpHecTROSS Combination pH Micro Electrode and an Orion Dual Star pH and ISEBenchtop Meter (Thermo Fisher Scientific, Waltham, Mass.). Tryptophanfluorescence spectra were recorded at each pH value and were analyzedusing an online protein fluorescence and structural toolkit (PFAST) toobtain the positions of spectral maxima (λ_(max)). Finally, thepositions of λ_(max) were plotted as a function of the various pHs andthe Henderson-Hasselbalch equation employed to fit the data:

${\lambda_{\max} = {\lambda_{\max}^{2} + \frac{\lambda_{\max}^{1} - \lambda_{\max}^{2}}{1 + {10^{n{({{pH} - {pK}})}}}}}},$

where λ_(max) ¹ and λ_(max) ² are the positions of spectral maxima atthe end and beginning of the State II-State III transition,respectively, n is the cooperativity parameter, and pK is the midpointof transition.

Results.

The NOTA (1,4,7-triazacyclononane-N′,N″,N′″-triacetic acid) chelator wasconjugated to various pHLIPs in two ways (FIGS. 2A-2C). The NOTA-pHLIPcompounds contain NOTA with three carboxylic acid groups conjugated tothe N-terminus of the peptide (FIG. 2A). The NO2A-cyspHLIP compoundscontain NO2A (1,4,7-Triazacyclononane-1,4-bis-aceticacid-7-maleimidoethylacetamide) with two carboxylic acid groups, wherethe third group was used to conjugate the chelator to the peptidethrough the cysteine side chain (FIG. 2B). The Cu²⁺ and AlF²⁺ NOTAcomplexes have an overall charge of −1 and the Ga³⁺ complexes have anoverall neutral charge for the chelator-metal complex with a neutralcharge at the N-terminus of the pHLIP. The Cu²⁺ and AlF²⁺ complexes withthe NO2A-chelates have an overall neutral charge and the Ga³⁺ complexeshave an overall charge of +1 for the chelator-metal complex with thepositive charge preserved at the N-terminus of the pHLIP. The fourpeptide sequences are listed in Table 1.

The ⁶⁴Cu radiolabeling of each of the conjugates was achieved withgreater than 60% (isolated) yield with greater than 93% radiochemicalpurity; the [¹⁸F]—AlF radiolabeling of each of the conjugates was 4-50%(isolated and decay corrected to start of synthesis) yield with greaterthan 95% radiochemical purity; the ⁶⁷Ga radiolabeling of each of theconjugates was achieved with greater than 70% (isolated) yield (relativeto the amount of ⁶⁷Ga was used to start the synthesis) with greater than95% radiochemical purity. Table 2 and FIG. 21 lists the specificactivity of each radiolabeled complex.

TABLE 2 Specific Activity Specific Activity Imaging Agent (μCi/nmol)^(a)(=82 Ci/nmol)^(b) ⁶⁴Cu-NOTA-Var7 82.4 N/A [¹⁸F] -A1F-NOTA-Var7 79.9 N/A⁶⁴Cu-NOTA-Var3 132.8^(c) N/A [¹⁸F] -A1F-NOTA-Var3 101.3 N/A ⁶⁴Cu-NOTA-WT116.4 N/A [¹⁸F] -A1F-NOTA-WT 135.9 N/A ⁶⁷Ga-NOTA-Var3 63.8-71.7 N/A⁶⁴Cu-NO2A-cysVar7 113.7 219.6 [¹⁸F]-A1F-NO2A-cysVar7 55.9 178.1⁶⁴Cu-NO2A-cysVar3 45.5-523.3 47.5-340.2 [¹⁸F]-A1F-NO2A-cysVar3 31.2-84.238.9-351.4 ⁶⁴Cu-NO2A-cysWT 215.6 99.1 [¹⁸F]-A1F-NO2A-cysWT 19.6 N/A⁶⁷Ga-NO2A-cysVar3 51.4-69.4 N/A ⁶⁸Ga-NO2A-cysVar3 94.5 N/A[¹⁸F]-A1F-NO2A-C²⁷Var3 85.2 ¹⁸7.6 ⁶⁴Cu-NO2A-C²⁷Var3 277.5 202.4⁶⁷Ga-NO2A-C²⁷Var3 284.0 N/A ^(a)Approximated from the amount of totalpeptide added. ^(b)Calculated from the UV/vis standard concentrationcurve. ^(c)In a separate experiment, a specific activity of 1656nCi/nmol was attained, but the lower specific activity material was usedin the studies in order to be comparable throughout.

Example 2: In Vitro and In Vivo Studies with the Complexes of thePresent Technology

This Example demonstrates that the complexes of the present technologyare useful in methods for detecting acidic diseased tissues (e.g., solidtumors) in a subject. FIGS. 22-71 further illustrate the advantages ofthe complexes of the present technology, including in comparison to,e.g., NOTA compounds.

Biophysical Studies.

Using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomesas model membranes, a comparative biophysical investigation of thepH-dependent interaction of NOTA-pHLIP and NO2A-cyspHLIP complexes withthe lipid bilayer indicated that NOTA complexes adopt unfavorableconformations at the membrane surface at physiological and low pHcompared to NO2A complexes. Although biophysical measurements aretypically conducted in solutions without the further addition of ions,the performance of these complexes both in the absence and presence ofphysiological levels of free magnesium and calcium ions wasinvestigated. Wild-type complexes appear to be sensitive to the presenceof divalent cations, most likely due to the multiple protonatableaspartic and glutamic acid residues at the inserting end of the peptide,which tend to form divalent complexes with these ions at physiologicalpH. Plots demonstrating the changes in fluorescence spectra ofAlF-NOTA-Var3, Cu-NOTA-Var3, AlF-NO2A-cysVar3, and Cu-NO2A-cysVar3 uponinteraction with membranes are shown in FIG. 9.

The ex vivo biodistribution data show that the metallated NOTA-pHLIPvariants are quickly cleared via both the hepatobiliary and renalpathways to reduce the overall circulating radioactivity significantlyby 12 h post injection. The rapid excretion of the ⁶⁴Cu-NOTA-pHLIPvariants from the intestines and slow elimination from the kidneys overtime indicated that the compounds were not being taken up by the cellswithin these organs, but were being eliminated via normal peptidemetabolic pathways.

Additionally, when comparing the 2 h ex vivo data for kidneys-to-largeintestine ratios [⁶⁴Cu-NOTA-WT is 0.35±0.11; [¹⁸F]—AlF-NOTA-WT is0.42±0.03; ⁶⁴Cu-NOTA-Var3 is 0.29±0.12; [¹⁸F]—AlF-NOTA-Var3 is0.52±0.15; ⁶⁴Cu-NOTA-Var7 is 0.32±0.07; [¹⁸F]—AlF-NOTA-Var7 is0.55±0.19], there was no significant difference between the excretionpathways of variants radiolabeled with ⁶⁴Cu or ¹⁸F. Without wishing tobe bound by theory, it is believed that the overall charge of thecomplexes may improve the elimination of these compounds, whiledecreasing the likelihood of uptake in low pH environments, such astumors, which may require more residence time in the blood. FIGS. 3A-3Cshow the lack of tumor targeting with NOTA-pHLIP complexes. This isconsistent with previous studies that described ⁶⁴Cu-NOTA-Var7, whichshowed in vivo localization that was inversely related to measuredextracellular pH (pH_(e)), but exhibited limited overall tumor uptake(1.36±0.43% ID/g). See Viola-Villegas et al., PNAS 111:7254-7259 (2014).

In contrast, the neutrally- and positively-charged NO2A complexes of thepresent technology conjugated to the cysteine residue at the N-terminalpart of the pHLIP (which preserve their positive charge at theN-terminus) show prolonged blood circulation with slower clearance,consequently leading to better perfusion of the complexes in the tumormicroenvironment. FIG. 3B and FIG. 3C show the biodistribution of ⁶⁴Cu-and ¹⁸F-NO2A complexes in selected tissues at 4 h and 24 h. The steadyincrease of the tumoral uptake of each of the NO2A complexes impliesslow localization in the tumor site. Var7 complexes exhibit the fastestblood clearance with the highest signal in the kidneys at 4 h p.i. andkidney clearance at later time points. The radiometallated NO2A-cysVar7complexes had the shortest blood retention with similar tumoral uptakeat 4 h compared to the other radiolabeled compounds.

This rapid blood clearance may make the radiometallated NO2A-cysVar7complexes useful in diagnostic imaging methods that entail shorterimaging times (tumor is visible in the slices at the tumor level inFIGS. 8A-8B) as long as the tumor is sufficiently distant from thekidneys (the major organ visible in the MIP in FIGS. 8A-8B). Withoutwishing to be bound by theory, it is believed that the rapid clearanceof the Var7 complexes may be a function of their physicalcharacteristics. While the Var7 complexes have insertion pKs that aresimilar to the NO2A-cysVar3 complexes, they are less hydrophobic thanthe NO2A-cysVar3 complexes, which may account for their faster bloodclearance properties (e.g., as indicated by lower amounts ofNO2A-cysVar7 complexes in the blood at every time point) andconsequently, their lower tumoral uptake.

NO2A-cysWT compounds exhibited highest accumulation in liver and spleencompared to NO2A-cysVar3 and NO2A-cysVar7. Both [¹⁸F]—AlF-NO2A-cysWT and⁶⁴Cu-NO2A-cysWT have slightly higher liver and spleen uptake compared tothe corresponding cysVar3 and cysVar7 conjugates. All of the NO2Acomplexes demonstrate a pH-dependent interaction with the lipid bilayerof the cellular membrane (see FIG. 10 and FIG. 11), but the NO2A-cysWTcomplexes exhibit the highest affinity to the lipid membrane atphysiological pH and slower rate of membrane insertion. Additionally,the pK of transition from the membrane-bound state to the inserted stateis higher for NO2A-cysWT complexes compared with NO2A-cysVar3 andNO2A-cysVar7 complexes. This shift in pK is most likely responsible forthe higher uptake of the NO2A-cysWT complexes by the liver and spleen.

The ⁶⁴Cu-NO2A-cys pHLIP complexes show decreased radioactivityaccumulation in the kidney from 4 h p.i. to 24 h p.i., which is due toclearance of the drug rather than uptake; whereas, the liver and spleenuptake remains similar for the two time points. The liver and spleenuptake may be due to loss of the ⁶⁴Cu from the chelator, digestion ofthe radiolabeled complex in vivo, or accumulation of intact radiolabeledcomplex in these organs.

While not wishing to be bound by theory, it is believed thatintroduction of a benzene ring in close proximity to the N-terminus ofthe peptide sequence and using a negatively charged metal complex shouldbe avoided in pHLIPs.

The ⁶⁴Cu-NO2A-cysVar3, [¹⁸F]—AlF-NO2A-cysVar3, ⁶⁷Ga-NO2A-cysVar3, and⁶⁷Ga-NO2A-C²⁷Var3 have the highest tumoral uptake, prolonged retentionin the tumor, and minimal accumulation in other organs based on the exvivo biodistribution data. FIGS. 4A-4B show the pH-dependent insertionand tabulates the percentage of the NO2A-cysVar3 complex population thatis inserted in the membrane at various pHs for the Cu- and F-labeledcomplexes. The results indicate that less than 5% of the NO2A-cysVar3complexes should be inserted into the membrane at pH 7.4 (physiologicalpH), whereas greater than 25% of the NO2A-cysVar3 complexes should beinserted into the cellular membrane at pH 6.0 (the approximate pH at thesurface of cancer cells). In addition, the membrane insertion of Var3and Var7 takes place at a rate two orders of magnitude higher than therate of insertion of WT.

In Vivo Studies in Prostate, Melanoma and Brain Tumor Models.

The ⁶⁴Cu- and [¹⁸F]—AlF-NO2A-cysVar3 compounds were compared in melanomaand prostate cancer models. FIG. 5 shows the in vivo PET images of theuptake of these two labelled compounds in tumors at 4 and 24 h p.i. Thegraph in FIG. 6 directly compares the tumoral uptake of these twolabelled compounds at 4 h, 6 h, and 24 h p.i. in four of the tumormodels investigated in this study. Due to the large variation in tumorsize for the LNCaP tumors, the ex vivo data shows a minimal increase intumoral uptake over the PC3 tumors, but both the murine B16-F10 melanomatumors and 4T1 breast cancer tumors showed greater uptake of the ⁶⁴Cu-and [¹⁸F]—AlF-NO2A-cysVar3 compounds compared to the human prostatecancer models. The greater uptake in the allografted tumors may indicatethat the relative overall tumor environment is less acidic (higher) inthe slower growing human cell lines investigated (PC3 and LNCaP), but ismore acidic (lower) in the faster growing murine cell lines (B16-F10 and4T1). ⁶⁴Cu-NO2A-cysVar3 and [¹⁸F]—AlF-NO2A-cysVar3 exhibited a hightumor:background contrast in B16-F10 murine melanoma tumor-bearing mice,which was useful to identify very small tumors (<17 mg; ˜4 mm³) in thePET image.

Because of the low uptake of the complexes of the present technology inbrain tissue, the [¹⁸F]—AlF-NO2A-cysVar3 was also evaluated in U87MGtumor-bearing mice. These tumors were implanted into the brain cavity orxenografted subcutaneously on the flank of nude mice. FIG. 12A shows theimaging of these tumors. The uptake in the U87MG tumors that wereimplanted into the brain cavity showed significant uptake of the tracerin the portion of the tumor that was outside the skull. During necropsy,these tumors did not show significant infiltration of the brain. The exvivo biodistribution of [¹⁸F]—AlF-NO2A-cysVar3 at 6 h p.i. in the nudemice with flank tumors is included in FIG. 12B. In order to conclusivelydetermine if the uptake of [¹⁸F]—AlF-NO2A-cysVar3 was only in theportion of the tumor outside of the skull, a group of mice with GSC 5-22tumors orthotopically xenografted behind an intact blood-brain barrier(BBB) were obtained. During necropsy, these tumors had infiltrated halfof the brain, but there was no uptake observed in the PET image. SeeFIG. 12A. For the brain tumor models, both an orthotopic and asubcutaneous (flank) brain tumor mouse model indicated that the[′⁸F]—AlF-NO2A-cysVar3 was unable to penetrate an intact BBB, but wasable to target brain tumors that had developed outside the BBB (FIG.12A). Thus, the complexes disclosed herein may be useful in identifyingmetastases and possibly brain tumors in situations of compromised BBBs.

Specific activity investigation. The complexes disclosed herein wereprepared to have comparable specific activities (approximately 100μCi/nmol (3.7 MBq/nmol)) for most of the studies. A few additionalstudies were conducted with higher and lower specific activities inorder to compare the effect of specific activity on tumoral uptake. Thedata sets were analyzed individually and showed that specific activitydid not impact the biodistribution of the tracers, especially tumoraluptake. Thus, all of the data points for similar tumor models wereaveraged in aggregate (regardless of specific activity) and tabulated inFIGS. 22-71.

Tissue Autoradiography Results.

The uptake of ⁶⁴Cu-NO2A-cysVar3 (24 h p.i.) in LNCaP and PC3 tumors wasinvestigated via autoradiography with viable tissue stained withhematoxylin and eosin (H&E) to determine if the uptake was specific toregions showing indications of metabolic stress or necrosis. FIG. 7shows that the uptake of ⁶⁴Cu-NO2A-cysVar3 appears to correlate in partwith necrotic regions in LNCaP tumors, but not in PC3 tumors. Withoutwishing to be bound by theory, it is believed that this observation maybe a result of the PC3 tumors having less overall necrosis than theLNCaP tumors in this study. Further, autoradiography result showsincreased relative uptake of ⁶⁴Cu-NO2A-cysVar3 (24 h p.i.) in LNCaP overPC3 tumors (FIG. 13). H&E staining of tumor sections from 4T1 tumorsexcised 5-12 days post inoculation showed nonnecrotic tumor tissue;thus, the greater tumoral uptake of the ⁶⁴Cu-NO2A-cysVar3 (according tothe ex vivo biodistribution data) does not appear to be correlated withnecrosis.

In Vivo Studies in 4T1 Tumor Models.

Direct in vivo evaluation of all twelve complexes disclosed herein wascarried out in 4T1 orthotopic (surgically implanted) allografted femaleBALB/c mice in order to determine which compounds would be translatedinto clinical studies. FIGS. 3A-3C show the in vivo PET imaging slicesat the level of the tumor at 4 h p.i. of eleven of the fifteen complexestested and the ex vivo biodistribution in selected organs at 4 and 24 hp.i. (% ID/g, mean±S.D.). The corresponding maximum intensityprojections (MIPs) from the PET imaging study and the PET imaging slicesat the level of the tumor with a lower maximum value for bettervisualization of the tumor at 4 h p.i. for the [¹⁸F]—AlF-NO2A-cyspHLIPcomplexes are shown in FIGS. 8A-8B.

The 4 h ex vivo biodistribution values (% ID/g, mean±S.D.) are collectedin Tables 3 and 4 for twelve of the radiopharmaceutical complexesdisclosed herein, and the complete biodistribution results for all timepoints of all fifteen complexes are tabulated in FIGS. 22-71.

Table 3 shows a biodistribution (% ID/g) of six radiolabeled N-terminusderivatized NOTA-pHLIP derivatives in 4T1 allografted female BALB/c miceat 4 h p.i.

TABLE 3 ⁶⁴CU- ⁶⁴Cu- ⁶⁴Cu- [¹⁸F]-A1F- [¹⁸F]-A1F- [¹⁸F]-A1F- NOTA-WTNOTA-Var3 NOTA-Var7 NOTA-WT NOTA-Var3 NOTA-Var7 Tissue (n = 4) (n = 5)(n = 4) (n = 5) (n = 4) (n = 5) Blood 1.192 ± 0.263 1.371 ± 0.244 0.833± 0.068 1.978 ± 0.192 2.201 ± 0.374 2.292 ± 0.277 Heart  0.50 ± 0.04 0.49 ± 0.05  0.35 ± 0.04  0.66 ± 0.12  0.78 ± 0.08  0.78 ± 0.06 Lungs1.414 ± 0.083 1.420 ± 0.117 0.996 ± 0.066 2.049 ± 0.211 2.217 ± 0.3802.325 ± 0.212 Liver 0.904 ± 0.097 1.034 ± 0.150 0.739 ± 0.041 0.973 ±0.213 0.923 ± 0.153 1.089 ± 0.066 Spleen 0.318 ± 0.023 0.340 ± 0.0320.279 ± 0.016 0.434 ± 0.058 0.469 ± 0.093 0.485 ± 0.029 Pancreas 0.261 ±0.066 0.279 ± 0.041 0.198 ± 0.043 0.324 ± 0.059 0.332 ± 0.020 0.373 ±0.051 Stomach 0.282 ± 0.080 0.194 ± 0.050 0.300 ± 0.076 0.518 ± 0.1520.482 ± 0.223 0.593 ± 0.153 S. intestine 0.391 ± 0.082 0.320 ± 0.0420.307 ± 0.077 0.820 ± 0.245 0.927 ± 0.152 0.776 ± 0.196 L. intestine 9.96 ± 3.42  7.45 ± 2.73  7.78 ± 0.61 24.06 ± 2.85 22.05 ± 2.46 22.08 ±6.02 Kidneys 6.232 ± 0.613 7.165 ± 1.126 6.790 ± 0.461 8.550 ± 1.1388.237 ± 1.106 7.759 ± 0.658 Muscle 0.166 ± 0.008 0.171 ± 0.017 0.118 ±0.025 0.283 ± 0.041 0.282 ± 0.055 0.325 ± 0.073 Bone 0.274 ± 0.051 0.339± 0.018 0.229 ± 0.093 0.671 ± 0.305 0.540 ± 0.119 0.571 ± 0.202 Skin0.715 ± 0.109 0.775 ± 0.102 0.563 ± 0.145 1.032 ± 0.133 1.126 ± 0.1151.377 ± 0.148 Brain 0.054 ± 0.002 0.063 ± 0.011 0.042 ± 0.004 0.100 ±0.015 0.107 ± 0.023 0.118 ± 0.011 Tumor 0.679 ± 0.025 0.649 ± 0.0910.546 ± 0.075 1.005 ± 0.232 0.923 ± 0.119 1.105 ± 0.125

Table 4 shows a biodistribution (% ID/g) of six radiolabeled cysteinederivatized NO2A-cyspHLIP derivatives in 4T1 allografted female BALB/cmice at 4 h p.i.

TABLE 4 ⁶⁴CU- ⁶⁴CU- ⁶⁴CU- [¹⁸F]-A1F- [¹⁸F]-A1F- [¹⁸F]-A1F- NO2A- NO2A-NO2A- NO2A- NO2A- NO2A- cysWT cysVar3 cysVar7 cysWT cysVar3 cysVar7Tissue (n = 4) (n = 5) (n = 4) (n = 4) (n = 10) (n = 4) Blood 17.6 ±0.86 15.4 ± 1.76 2.31 ± 0.18 15.9 ± 0.63 20.8 ± 1.88 1.64 ± 0.18 Heart5.52 ± 0.15 5.12 ± 0.63 2.68 ± 0.33 4.78 ± 0.53 6.29 ± 1.03 0.89 ± 0.12Lungs 12.3 ± 1.55 14.7 ± 0.93 4.21 ± 0.23 8.73 ± 0.84 11.8 ± 3.79 1.66 ±0.07 Liver 21.9 ± 0.64 10.4 ± 0.63 15.6 ± 1.58 12.6 ± 11.4 9.06 ± 1.242.90 ± 0.18 Spleen 7.07 ± 0.44 3.45 ± 0.35 3.31 ± 0.24 5.31 ± 0.34 3.73± 0.66 1.19 ± 0.10 Pancreas 3.00 ± 0.08 2.31 ± 0.25 1.84 ± 0.10 2.10 ±0.43 2.44 ± 0.20 0.49 ± 0.03 Stomach 1.82 ± 0.13 1.27 ± 0.35 3.33 ± 0.311.31 ± 0.96 1.29 ± 0.62 0.68 ± 0.10 S. intestine 4.56 ± 0.26 2.68 ± 0.246.37 ± 0.11 2.85 ± 0.31 2.06 ± 0.28 0.91 ± 0.03 L. intestine 4.09 ± 0.342.99 ± 0.20 8.19 ± 0.85 1.47 ± 0.14 2.07 ± 0.63 0.91 ± 0.12 Kidneys 40.6± 4.04 24.0 ± 2.78 95.5 ± 11.9 59.1 ± 3.63 34.3 ± 6.91 2.46 ± 25.6Muscle 1.15 ± 0.08 1.10 ± 0.15 0.64 ± 0.07 1.23 ± 0.17 1.55 ± 0.27 0.35± 0.06 Bone 3.04 ± 0.11 1.10 ± 0.14 1.57 ± 0.18 1.94 ± 0.45 1.66 ± 0.610.60 ± 0.12 Skin 3.23 ± 0.12 3.43 ± 0.34 2.59 ± 0.05 2.85 ± 0.22 2.92 ±0.71 1.59 ± 0.10 Brain 0.62 ± 0.06 0.35 ± 0.07 0.25 ± 0.02 0.34 ± 0.060.52 ± 0.14 0.05 ± 0.01 Tumor 11.7 ± 1.71 8.21 ± 0.86 9.07 ± 1.81 8.16 ±0.53 10.6 ± 2.26 8.61 ± 1.21

Two positron emitting radionuclides (⁶⁴Cu and ¹⁸F) were used to labelthe NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivobiodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. Allof the complexes were radiolabeled with ⁶⁴Cu or [¹⁸F]—AlF in good yield.The in vivo biodistribution of the twelve complexes in 4T1 orthotopicallografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeledwith either ¹⁸F (4T1 uptake; 8.9±1.7% ID/g at 4 h p.i.), ⁶⁴Cu (4T1uptake; 8.2±0.9% ID/g at 4 h p.i. and 19.2±1.8% Dig at 24 h p.i.),⁶⁷Ga-NO2A-cysVar3 (4T1 uptake; 12±3% ID/g at 4 h p.i. and 26±4% ID/g at24 h p.i.), or ⁶⁷Ga-NO2A-C²⁷Var3 (4T1 uptake; 20.5±1.0% ID/g at 4 h p.i.and 23±5% ID/g at 24 h p.i.) show superior biodistribution properties(FIGS. 16 and 17).

⁶⁷Ga-NO2A-cysVar3 (positively charged metal complex and positivelycharged N-terminus) shows a slightly higher tumoral uptake than⁶⁴Cu-NO2A-cysVar3 (neutrally charged metal complex and positivelycharged N-terminus) at 4 and 24 h.p.i. See FIGS. 16 and 18. However,this was not true for the ⁶⁸Ga-NO2A-cysVar3 complex (FIG. 16), whichsuggests a difference in the complex formation between the differentradiolabels and pHLIP complex. Further, ⁶⁷Ga-NO2A-C²⁷Var3 exhibitedhigher tumor accumulation at 4 h (see FIGS. 16 and 19B) than⁶⁸Ga-NO2A-cysVar3 complex.

NO2A-C²⁷Var3 had much higher kidney uptake compared to the NO2A-C²Var3complexes (compare FIG. 17 and Table 4) with consistent tumoral uptake(approximately 10% ID/g) at 4, 6, and 24 h post-injection for theneutral complexes (e.g., ¹⁸F and ⁶⁴Cu). The ⁶⁴Cu-NO2A-C²⁷Var3 alsoshowed higher uptake in the small and large intestines at longer timepoints, which may make imaging with this complex difficult at longertime points (FIG. 19A). Interestingly, ⁶⁷Ga-NO2A-C²⁷Var3 showed rapidand prolonged tumoral uptake (approximately 20% ID/g) by 4 hpost-injection, with low background (non-target tissues not includingthe kidneys), which allowed visualization of the tumor (FIG. 19B).

The SPECT/CT imaging with ⁶⁷Ga-NO2A-cysVar3 and PET images of the⁶⁸Ga-NO2A-cysVar3 radioconjugates are shown in FIG. 18 and FIG. 20,respectively. The ex vivo biodistribution showed that the tumoral uptake(at 4 h p.i.) was 12±3% ID/g for ⁶⁷Ga-NO2A-cysVar3, but 6±2% ID/g for⁶⁸Ga-NO2A-cysVar3 and 2.05±0.17% ID/g for the ⁶⁷Ga-NOTA-Var3. Thetumoral uptake (at 24 h p.i.) of the ⁶⁷Ga-NO2A-cysVar3 in the imagingstudy (500 μCi injected) was 34±3% ID/g compared to 26±4% ID/g from thebiodistribution study (50 μCi injected), but was greater than the2.93±0.11% ID/g observed with ⁶⁷Ga-NOTA-Var3. In both imagingexperiments, the tumor was detectable, but more kidney uptake wasobserved relative to the ⁶⁴Cu and Al¹⁸F compounds. Additionally, the 4 hPET image shows significant blood pool, which is greatly reduced in the24 h SPECT images. These results indicate that the positive charge ofthe metal-chelate complex shifts the excretion of the radiotracer towardkidney uptake, but does not decrease the tumoral uptake in theNO2A-cysVar3 radioconjugates. The radioconjugate of NOTA-Var3 showedlimited tumor targeting abilities, despite having a neutralmetal-chelator complex. Additionally, the PET and SPECT/CT imaging ofthe radiopharmaceutical complexes utilizing the NO2A-C²⁷Var3 areincluded in FIGS. 19A and 19B.

These data demonstrate that all complexes with the NO2A chelatoroutperform the complexes with the NOTA chelator. It appears thatNO2A-cyspHLIP complexes have a longer blood half-life, which results inhigher targeting and longer retention within the tumor while exhibitingrapid clearance from nontarget tissues and the blood. Conversely, theNO2A-C²⁷Var3 compounds appear to have shorter blood half-lives than theNO2A-C²Var3 compounds with greater kidney uptake and retention. The⁶⁷Ga-NO2A-C²⁷Var3 rapidly targets the tumor (20.5±1.0% ID/g) by 4 h p.i.and remains relatively constant out to 24 h p.i. (23±5% ID/g). The rapidaccumulation within the tumor indicates that the NO2A-C²⁷Var3 complexmay be useful for PET imaging with the shorter-lived ⁶⁸Ga.

Taken together, the results demonstrate that ⁶⁴Cu-NO2A-cysVar3,[¹⁸F]—AlF-NO2A-cysVar3, ⁶⁷Ga-NO2A-cysVar3, and ⁶⁷Ga-NO2A-C²⁷Var3 showedthe greatest tumoral uptake and significant tumor-to-background contrastin the imaging studies described herein. The in vivo properties of⁶⁴Cu-NO2A-cysVar3 and [¹⁸F]—AlF-NO2A-cysVar3 were investigated in PC3and LNCaP tumor-bearing mice to compare the tumoral uptake observed inprevious studies. In these tumor models, the tumor accumulation of⁶⁴Cu-NO2A-cysVar3 and [¹⁸F]—AlF-NO2A-cysVar3 was significantly highercompared to previous generations of PET isotope-labeled complexes,especially at 24 h p.i. (FIGS. 14A-14B).

Comparing the tumor:tissue ratios with the previously reported compounds¹⁸F-py-click-6Ahx-WT and ⁶⁸Ga-DO3A-cysVar7, the tumor:muscle ratio at 4h p.i. were 7.4±1.3 (⁶⁴Cu-NO2A-cysVar3), 6.9±1.9([¹⁸F]—AlF-NO2A-cysVar3), 4±2 (18F-py-click-6Ahx-WT), and 1.1±1.5(⁶⁸Ga-DO3A-cysVar7) in PC3 tumor-bearing male nude mice; the tumor:boneratios were 2.7±0.6 (⁶⁴Cu-NO2A-cysVar3), 3.2±0.7([¹⁸F]—AlF-NO2A-cysVar3), 1.3±0.4 (¹⁸F-py-click-6Ahx-WT), and 0.4±0.3(⁶⁸Ga-DO3A-cysVar7) in PC3 tumor-bearing male nude mice. See FIGS.15A-15B. Additionally, as shown in FIG. 15C, the tumor:muscle ratios inLNCaP tumor-bearing male nude mice at 4 h p.i. were 6±2(⁶⁴Cu-NO2A-cysVar3), 4.7±0.9 ([¹⁸F]—AlF-NO2A-cysVar3), and 6±3(¹⁸F-py-click-6Ahx-WT); the tumor:bone ratios in LNCaP tumor-bearingmale nude mice at 4 h p.i. were 4.2±1.7 (⁶⁴Cu-NO2A-cysVar3), 4.3±1.1([¹⁸F]—AlF-NO2A-cysVar3), and 1.8±0.6 (¹⁸F-py-click-6Ahx-WT).

The tumor targeting ratios of ⁶⁴Cu-NO2A-cysVar3 and[¹⁸F]—AlF-NO2A-cysVar3 were superior to that observed with⁶⁸Ga-DO3A-cysVar7. While the ¹⁸F-py-click-6Ahx-WT had similartumor:muscle ratios, the tumor:bone ratios were much lower than⁶⁴Cu-NO2A-cysVar3 and [¹⁸F]—AlF-NO2A-cysVar3 in both prostate cancertumor models. The decreased tumor:bone ratios may indicate that thepreviously reported ¹⁸F-py-click-6Ahx-WT was more prone todefluorination than the ¹⁸F-labeled complexes of the present technology.Further, unlike ¹⁸F-py-click-6Ahx-WT, the complexes of the presenttechnology do not require HPLC purification or heating beyond 80° C.,which is optimal for rapid dose-on-demand production of the radiolabeledcomplexes for patient trials.

These results demonstrate that the complexes of the present technologyare useful in methods for detecting solid tumors in a subject.Accordingly, the compounds and complexes disclosed herein are useful inmethods for detecting acidic diseased tissues in a subject.

Example 3: Use of the Complexes of the Present Technology to DetectAcidic Diseased Tissues in a Subject

This Example demonstrates that the complexes of the present technologyare useful in methods for detecting acidic diseased tissues in asubject.

Cerebral ischemia is induced by occlusion of the right middle cerebralartery for 30 min. Wild-type (WT) mice will be given a complex of thepresent technology (500-600 μCi (9-11 nmol) for in vivo imaging) at 0,6, 24 and 48 h after ischemia. Mice will be sacrificed at no later than48 hours after receiving the complex. PET/CT (2 and 4 h p.i.) andSPECT/CT (6 and 24 h p.i.) imaging studies will be carried out.

It is anticipated that the complexes disclosed herein will localize tothe acidic diseased brain tissue (tissue impacted by ischemia), withminimal accumulation in non-target tissues.

These results demonstrate that the complexes of the present technologyare useful in methods for detecting acidic diseased tissues in asubject.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds, compositions, or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso form part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments (for example, journals, articles and/or textbooks) referred toin this specification are herein incorporated by reference as if eachindividual publication, patent application, issued patent, or otherdocument was specifically and individually indicated to be incorporatedby reference in its entirety. Definitions that are contained in textincorporated by reference are excluded to the extent that theycontradict definitions in this disclosure.

The present technology may include, but is not limited to, the featuresand combinations of features recited in the following letteredparagraphs, it being understood that the following paragraphs should notbe interpreted as limiting the scope of the claims as appended hereto ormandating that all such features must necessarily be included in suchclaims:

-   A. A compound or pharmaceutically acceptable salt thereof comprising    -   a pH (low) insertion peptide (“pHLIP”) configured to localize to        an extracellular environment having a pH that is lower than 7.4,        wherein the pHLIP comprises a C-terminus and an N-terminus; and    -   X¹ covalently attached to a heteroatom of a side chain of an        amino acid residue of the pHLIP, where the amino acid residue is        from 0, 1, 2, 3, 4, 5, or 6 residues from the C-terminus or the        N-terminus;    -   wherein        -   X¹ is of Formula I

-   -   -   where Z¹ and Z² are each independently a lone pair of            electrons (i.e. providing an oxygen anion) or H; and        -   Z³ is —NH-alkylene-, —NH—CH₂CH₂—((poly(alkylene glycol))-,

-   -   -    where n is 1, 2, 3, 4, 5, 6, 7, or 8, or a bond.

-   B. The compound of Paragraph A, wherein the pHLIP is configured to    localize to an extracellular environment having a pH that is lower    than 7.1.

-   C. The compound of Paragraph A or Paragraph B, wherein the pHLIP is    configured to localize to a tissue comprising the extracellular    environment.

-   D. The compound of any one of Paragraphs A-C, wherein Z³ is    —NH—(CH₂)_(m)—, —NH—CH₂CH₂—(O—CH₂CH₂)_(p)—,

-    or a bond, where    -   n is 1, 2, 3, 4, 5, 6, 7, or 8;    -   m is 1, 2, 3, 4, 5, 6, 7, or 8; and    -   p is 1, 2, 3, 4, 5, 6, 7, or 8.-   E. The compound of any one of Paragraphs A-D, wherein the pHLIP is

(SEQ ID NO: 1) ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT, (SEQ ID NO: 2)ACDDQNPWRAYLDLLFPTDTLLLDLLW, (SEQ ID NO: 3)ADDQNPWRAYLDLLFPTDTLLLDLLWCG, or (SEQ ID NO: 4)ACEEQNPWARYLEWLFPTETLLLEL.

-   F. The compound of any one of Paragraphs A-E, wherein the amino acid    residue is a cysteine or lysine.-   G. The compound of any one of Paragraphs A-F, wherein X¹ is    covalently attached to a sulfur atom of a cysteine residue of the    pHLIP or is covalently attached to a c-nitrogen atom of a lysine    residue of the pHLIP.-   H. The compound of any one of Paragraphs A-G, wherein the compound    is AC(X¹)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO:5),    AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),    ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), or    AC(X¹)EEQNPWARYLEWLFPTETLLLEL (SEQ ID NO:8), or a pharmaceutically    acceptable salt thereof-   I. The compound of any one of Paragraphs A-H, wherein X¹ is of    Formula Ia

-   J. The compound of any one of Paragraphs A-I, wherein the compound    is AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),    ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), or a    pharmaceutically acceptable salt thereof; wherein X¹ is of Formula    Ia.-   K. A complex comprising the compound of any one of Paragraphs A-J    and a radionuclide.-   L. The complex of Paragraph K, wherein X¹ is of Formula II

-    wherein M¹ is ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺,    ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.-   M. The complex of Paragraph K or Paragraph L, wherein X¹ is of    Formula IIa

-   N. The complex of any one of Paragraphs K-M, wherein the complex is    AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),    ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), or a    pharmaceutically acceptable salt thereof; wherein X¹ is of Formula    IIa.-   O. A composition comprising a complex of any one of Paragraphs K-N    and a pharmaceutically acceptable carrier.-   P. A pharmaceutical composition for detecting a tissue comprising an    extracellular environment having a pH that is lower than 7.4, the    pharmaceutical composition comprising an effective amount of the    complex of any one of Paragraphs K-N and a pharmaceutically    acceptable carrier.-   Q. The pharmaceutical composition of Paragraph P, wherein the tissue    comprises one or more of the group consisting of an atherosclerotic    plaques, ischemic myocardium, a tissue impacted by stroke, and a    cancer tissue.-   R. The pharmaceutical composition of Paragraph Q, wherein the cancer    tissue is selected from the group consisting of breast cancer,    colorectal cancer, cervical cancer, ovarian cancer, liver cancer,    bladder cancer, hepatoma, hepatocellular carcinoma, brain tumors,    lung cancer, gastric or stomach cancer, pancreatic cancer, thyroid    cancer, kidney or renal cancer, prostate cancer, melanoma, sarcomas,    carcinomas, Wilms tumor, endometrial cancer, glioblastoma, squamous    cell cancer, astrocytomas, salivary gland carcinoma, vulvar cancer,    penile carcinoma, and head-and-neck cancer, preferably from the    group consisting of breast cancer, a brain tumor, prostate cancer,    melanoma, and a metastatic cancer thereof.-   S. A method for detecting solid tumors in a subject in need thereof    comprising    -   (a) administering an effective amount of a complex of any one of        Paragraphs K-N to the subject; and    -   (b) detecting the presence of a tissue comprising an        extracellular environment having a pH that is lower than 7.4 in        the subject by detecting radioactive levels emitted by the        complex that are higher than a reference value.-   T. The method of Paragraph S, wherein the radioactive levels emitted    by the complex are detected using positron emission tomography or    single photon emission computed tomography.-   U. The method of Paragraph S or Paragraph T, wherein the subject is    diagnosed with, or is suspected of having an atherosclerotic plaque,    ischemic myocardium, a tissue impacted by stroke, or cancer.-   V. The method of Paragraph U, wherein the cancer is selected from    the group consisting of breast cancer, colorectal cancer, cervical    cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,    hepatocellular carcinoma, brain tumors, lung cancer, gastric or    stomach cancer, pancreatic cancer, thyroid cancer, kidney or renal    cancer, prostate cancer, melanoma, sarcomas, carcinomas, Wilms    tumor, endometrial cancer, glioblastoma, squamous cell cancer,    astrocytomas, salivary gland carcinoma, vulvar cancer, penile    carcinoma, and head-and-neck cancer, preferably from the group    consisting of breast cancer, a brain tumor, prostate cancer,    melanoma, and a metastatic cancer thereof.-   W. The method of any one of Paragraphs S-V, wherein the complex is    administered into the cerebral spinal fluid or blood of the subject.-   X. The method of any one of Paragraphs S-W, wherein the complex is    administered intravenously, intramuscularly, intraarterially,    intrathecally, intracapsularly, intradermally, intraperitoneally,    intralesionally, transtracheally, subcutaneously,    intracerebroventricularly, orally, intranasally, rectally,    topically, or via inhalation.-   Y. The method of any one of Paragraphs S-X, wherein the radioactive    levels emitted by the complex are detected between 4 to 24 hours    after the complex is administered.-   Z. The method of any one of Paragraphs S-Y, wherein the radioactive    levels emitted by the complex are expressed as the percentage    injected dose per gram tissue (% ID/g).-   AA. The method of any one of Paragraphs S-Z, wherein the ratio of    radioactive levels between a tumor and normal tissue is about 2:1,    3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1,    35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1,    90:1, 95:1 or 100:1.-   AB. A kit comprising a compound of any one of Paragraphs A-J, at    least a radionuclide, and instructions for use.-   AC. The kit of Paragraph AB, wherein the radionuclide comprises is    ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺,    ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.

Other embodiments are set forth in the following claims, along with thefull scope of equivalents to which such claims are entitled.

1. A compound or pharmaceutically acceptable salt thereof comprising apH (low) insertion peptide (“pHLIP”) configured to localize to anextracellular environment having a pH that is lower than 7.4, whereinthe pHLIP comprises a C-terminus and an N-terminus; and X¹ covalentlyattached to a heteroatom of a side chain of an amino acid residue of thepHLIP, where the amino acid residue is from 0, 1, 2, 3, 4, 5, or 6residues from the C-terminus or the N-terminus; wherein X¹ is of FormulaI

where Z¹ and Z² are each independently a lone pair of electrons (i.e.,providing an oxygen anion) or H; and Z³ is

 where n is 1, 2, 3, 4, 5, 6, 7, or 8, —NH-alkylene-,—NH—CH₂CH₂-((poly(alkylene glycol))-, or a bond.
 2. (canceled) 3.(canceled)
 4. The compound of claim 1, wherein Z³ is

—NH—(CH₂)_(m)—, —NH—CH₂CH₂—(O—CH₂CH₂)_(p)—, or a bond, where n is 1, 2,3, 4, 5, 6, 7, or 8; m is 1, 2, 3, 4, 5, 6, 7, or 8; and p is 1, 2, 3,4, 5, 6, 7, or
 8. 5. The compound of claim 1, wherein the pHLIP is(SEQ ID NO: 1) ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT, (SEQ ID NO: 2)ACDDQNPWRAYLDLLFPTDTLLLDLLW, (SEQ ID NO: 3)ADDQNPWRAYLDLLFPTDTLLLDLLWCG, or (SEQ ID NO: 4)ACEEQNPWARYLEWLFPTETLLLEL.


6. The compound of claim 1, wherein the amino acid residue is a cysteineor lysine.
 7. The compound of claim 1, wherein X¹ is covalently attachedto a sulfur atom of a cysteine residue of the pHLIP or is covalentlyattached to a ε-nitrogen atom of a lysine residue the pHLIP.
 8. Thecompound of claim 1, wherein the compound isAC(X¹)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO:5),AC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), orAC(X¹)EEQNPWARYLEWLFPTETLLLEL (SEQ ID NO:8), or a pharmaceuticallyacceptable salt thereof.
 9. The compound of claim 1, wherein X¹ is ofFormula Ia


10. The compound of claim 1, wherein the compound isAC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), or a pharmaceuticallyacceptable salt thereof; wherein X¹ is of Formula Ia.
 11. A complexcomprising the compound of claim 1 and a radionuclide.
 12. The complexof claim 11, wherein X¹ is of Formula II

wherein M¹ is ⁶⁰Cu²⁺, ⁶¹Cu²⁺, ⁶²Cu²⁺, ⁶⁴Cu²⁺, ⁶⁷Cu²⁺, [¹⁸F]—AlF²⁺,⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, or ⁷¹Ga³⁺.
 13. The complex of claim 11, whereinX¹ is of Formula IIa


14. The complex of claim 11, wherein the complex isAC(X¹)DDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:6),ADDQNPWRAYLDLLFPTDTLLLDLLWC(X¹)G (SEQ ID NO:7), or a pharmaceuticallyacceptable salt thereof; wherein X¹ is of Formula IIa.
 15. A compositioncomprising a complex of claim 11 and a pharmaceutically acceptablecarrier.
 16. A pharmaceutical composition for detecting a tissuecomprising an extracellular environment having a pH that is lower than7.4, the pharmaceutical composition comprising an effective amount ofthe complex of claim 11 and a pharmaceutically acceptable carrier. 17.The pharmaceutical composition of claim 16, wherein the tissue comprisesone or more of the group consisting of an atherosclerotic plaque,ischemic myocardium, a tissue impacted by stroke, and a cancer tissue.18. (canceled)
 19. A method for detecting solid tumors in a subject inneed thereof comprising (a) administering an effective amount of acomplex of claim 11 to the subject; and (b) detecting the presence of atissue comprising an extracellular environment having a pH that is lowerthan 7.4 in the subject by detecting radioactive levels emitted by thecomplex that are higher than a reference value.
 20. The method of claim19, wherein the radioactive levels emitted by the complex are detectedusing positron emission tomography or single photon emission computedtomography.
 21. The method of claim 19, wherein the subject is diagnosedwith, or is suspected of having an atherosclerotic plaque, ischemicmyocardium, a tissue impacted by stroke, or cancer.
 22. (canceled) 23.(canceled)
 24. (canceled)
 25. The method of claim 19, wherein theradioactive levels emitted by the complex are detected between 4 to 24hours after the complex is administered.
 26. (canceled)
 27. (canceled)28. A kit comprising a compound of claim 1, at least a radionuclide, andinstructions for use.
 29. (canceled)